DHPLC PrinciplesAn Introduction to

Denaturing High Performance Liquid Chromatography

Pooria GillPhD of Nanobiotechnology

pooriagillyahoocom

In The Name of Allah

Nucleic Acids

bull DNAndash Linear

bull Nature-madendash High Molecular

Weightndash Low Molecular

Weightbull Man-made

ndash High Molecular Weight

ndash Low Molecular Weight

ndash Circularbull Nature-made

ndash High Molecular Weight

ndash Low Molecular Weight

bull Man-madendash High Molecular

Weightndash Low Molecular

Weight

bull RNAbull Nature-made

ndash High Molecular Weight

ndash Low Molecular Weight

bull Man-madendash High Molecular

Weightndash Low Molecular

Weight

Nucleic Acids OMICS

bull Genomicsndash Various analyses of DNAs

bull Qualitativebull Quantitative

bull Transcriptomicsndash Various analyses of RNAs

bull Qualitativebull Quantitative

Genomics Variations as MutationPolymorphism

bull Scanning Procedures for Unknown Mutation Detections those simple methods which rely on differences in electrophoretic properties being generated between mutant and wild-type nucleic acid by point mutations (these methods cannot as currently used detect all mutations do not localize them within the fragment and can only be applied to DNA fragments hundreds of bases long)

bull Screening Procedures for Known Mutation Detections those group which have the potential to detect all mutations Sequencing is more frequently used to detect unknown mutations than it is for diagnostic purposes

The Principles for Gene Variation Analysis

bull Bioinformatics

bull Biothermodynamics Biophysical-Chemistry

bull Spectroscopics

bull Electrophoretics

bull Chromatographics

Scanning Procedures

ndash Ribonuclease cleavage (RNAase)

ndash Denaturing gradient-gel electrophoresis

(DGGE) and related techniques

ndash Carbodiimide modification (CDI)

ndash Chemical cleavage of mismatch (CCM)

ndash Single-strand conformation polymorphism

(SSCP)

ndash Heteroduplex analysis (HET)

ndash Direct sequencing (DS)

Scanning Procedures

RGH Cotton Mutation Research 285 (1993) 125-

144

Screening Procedures

bull Allele-specific oligonucleotide (ASO)

bull Allele-specific amplification (ASA)

bull Ligation (LIG)

bull Primer extension (PEX)

bull Artificial introduction of restriction sites

(AIRS)

Screening Procedures

RGH Cotton Mutation Research 285 (1993) 125-144

Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as

heteroduplexes on hydrolink gels Trends Genet 7 5

bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels

bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity

bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description

bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)

Technological Improvement of HET to eg DHPLC

bull Patent Information1 Column matrix

ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria

ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236

ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA

DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex

Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]

ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]

ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA

ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]

ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]

Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing

ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]

ndash Licensed to Gene Machines Redwood City CA USA

DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific

Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]

Principle of DHPLC

Rapid denaturation of DNA by heating re-annealing by slow cooling

Heteroduplexes form in the presence of two different alleles

DHPLCGraphical Scheme

Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)

Nucleic Acid Amplifications eg PCR

PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System

from Invitrogen

ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture

composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D

polymerase Pyrococcus species GB-D polymerase possesses a proofreading

ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading

enzyme with Taq DNA polymerase at an optimized ratio increases fidelity

approximately eight times over that of Taq DNA polymerase alone and allows

amplification of simple and complex DNA templates The enzyme mixture is

provided with an optimized buffer that improves enzyme fidelity

ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer

formulation have been optimized for use with denaturing high-performance

liquid chromatography (dHPLC) systems They were developed and tested using

the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is

supplied at 1 unit per microl

Equipments

1 Thermocycler (with heated lid)

2 DHPLC WAVE DNA fragment analysis system

(Transgenomic)

3 DNA Sep HT cartridge (cat no DNA-99-3710

Transgenomic)

4 Transmit silicone sealing mats (see Note 2) (cat no

172024 Transgenomic)

5 PCR plates (see Note 3) (cat no 0030 127307

Eppendorf)

6 Chemical waste container

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Nucleic Acids OMICS

bull Genomicsndash Various analyses of DNAs

bull Qualitativebull Quantitative

bull Transcriptomicsndash Various analyses of RNAs

bull Qualitativebull Quantitative

Genomics Variations as MutationPolymorphism

bull Scanning Procedures for Unknown Mutation Detections those simple methods which rely on differences in electrophoretic properties being generated between mutant and wild-type nucleic acid by point mutations (these methods cannot as currently used detect all mutations do not localize them within the fragment and can only be applied to DNA fragments hundreds of bases long)

bull Screening Procedures for Known Mutation Detections those group which have the potential to detect all mutations Sequencing is more frequently used to detect unknown mutations than it is for diagnostic purposes

The Principles for Gene Variation Analysis

bull Bioinformatics

bull Biothermodynamics Biophysical-Chemistry

bull Spectroscopics

bull Electrophoretics

bull Chromatographics

Scanning Procedures

ndash Ribonuclease cleavage (RNAase)

ndash Denaturing gradient-gel electrophoresis

(DGGE) and related techniques

ndash Carbodiimide modification (CDI)

ndash Chemical cleavage of mismatch (CCM)

ndash Single-strand conformation polymorphism

(SSCP)

ndash Heteroduplex analysis (HET)

ndash Direct sequencing (DS)

Scanning Procedures

RGH Cotton Mutation Research 285 (1993) 125-

144

Screening Procedures

bull Allele-specific oligonucleotide (ASO)

bull Allele-specific amplification (ASA)

bull Ligation (LIG)

bull Primer extension (PEX)

bull Artificial introduction of restriction sites

(AIRS)

Screening Procedures

RGH Cotton Mutation Research 285 (1993) 125-144

Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as

heteroduplexes on hydrolink gels Trends Genet 7 5

bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels

bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity

bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description

bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)

Technological Improvement of HET to eg DHPLC

bull Patent Information1 Column matrix

ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria

ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236

ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA

DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex

Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]

ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]

ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA

ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]

ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]

Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing

ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]

ndash Licensed to Gene Machines Redwood City CA USA

DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific

Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]

Principle of DHPLC

Rapid denaturation of DNA by heating re-annealing by slow cooling

Heteroduplexes form in the presence of two different alleles

DHPLCGraphical Scheme

Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)

Nucleic Acid Amplifications eg PCR

PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System

from Invitrogen

ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture

composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D

polymerase Pyrococcus species GB-D polymerase possesses a proofreading

ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading

enzyme with Taq DNA polymerase at an optimized ratio increases fidelity

approximately eight times over that of Taq DNA polymerase alone and allows

amplification of simple and complex DNA templates The enzyme mixture is

provided with an optimized buffer that improves enzyme fidelity

ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer

formulation have been optimized for use with denaturing high-performance

liquid chromatography (dHPLC) systems They were developed and tested using

the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is

supplied at 1 unit per microl

Equipments

1 Thermocycler (with heated lid)

2 DHPLC WAVE DNA fragment analysis system

(Transgenomic)

3 DNA Sep HT cartridge (cat no DNA-99-3710

Transgenomic)

4 Transmit silicone sealing mats (see Note 2) (cat no

172024 Transgenomic)

5 PCR plates (see Note 3) (cat no 0030 127307

Eppendorf)

6 Chemical waste container

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Genomics Variations as MutationPolymorphism

bull Scanning Procedures for Unknown Mutation Detections those simple methods which rely on differences in electrophoretic properties being generated between mutant and wild-type nucleic acid by point mutations (these methods cannot as currently used detect all mutations do not localize them within the fragment and can only be applied to DNA fragments hundreds of bases long)

bull Screening Procedures for Known Mutation Detections those group which have the potential to detect all mutations Sequencing is more frequently used to detect unknown mutations than it is for diagnostic purposes

The Principles for Gene Variation Analysis

bull Bioinformatics

bull Biothermodynamics Biophysical-Chemistry

bull Spectroscopics

bull Electrophoretics

bull Chromatographics

Scanning Procedures

ndash Ribonuclease cleavage (RNAase)

ndash Denaturing gradient-gel electrophoresis

(DGGE) and related techniques

ndash Carbodiimide modification (CDI)

ndash Chemical cleavage of mismatch (CCM)

ndash Single-strand conformation polymorphism

(SSCP)

ndash Heteroduplex analysis (HET)

ndash Direct sequencing (DS)

Scanning Procedures

RGH Cotton Mutation Research 285 (1993) 125-

144

Screening Procedures

bull Allele-specific oligonucleotide (ASO)

bull Allele-specific amplification (ASA)

bull Ligation (LIG)

bull Primer extension (PEX)

bull Artificial introduction of restriction sites

(AIRS)

Screening Procedures

RGH Cotton Mutation Research 285 (1993) 125-144

Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as

heteroduplexes on hydrolink gels Trends Genet 7 5

bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels

bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity

bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description

bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)

Technological Improvement of HET to eg DHPLC

bull Patent Information1 Column matrix

ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria

ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236

ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA

DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex

Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]

ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]

ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA

ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]

ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]

Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing

ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]

ndash Licensed to Gene Machines Redwood City CA USA

DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific

Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]

Principle of DHPLC

Rapid denaturation of DNA by heating re-annealing by slow cooling

Heteroduplexes form in the presence of two different alleles

DHPLCGraphical Scheme

Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)

Nucleic Acid Amplifications eg PCR

PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System

from Invitrogen

ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture

composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D

polymerase Pyrococcus species GB-D polymerase possesses a proofreading

ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading

enzyme with Taq DNA polymerase at an optimized ratio increases fidelity

approximately eight times over that of Taq DNA polymerase alone and allows

amplification of simple and complex DNA templates The enzyme mixture is

provided with an optimized buffer that improves enzyme fidelity

ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer

formulation have been optimized for use with denaturing high-performance

liquid chromatography (dHPLC) systems They were developed and tested using

the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is

supplied at 1 unit per microl

Equipments

1 Thermocycler (with heated lid)

2 DHPLC WAVE DNA fragment analysis system

(Transgenomic)

3 DNA Sep HT cartridge (cat no DNA-99-3710

Transgenomic)

4 Transmit silicone sealing mats (see Note 2) (cat no

172024 Transgenomic)

5 PCR plates (see Note 3) (cat no 0030 127307

Eppendorf)

6 Chemical waste container

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

The Principles for Gene Variation Analysis

bull Bioinformatics

bull Biothermodynamics Biophysical-Chemistry

bull Spectroscopics

bull Electrophoretics

bull Chromatographics

Scanning Procedures

ndash Ribonuclease cleavage (RNAase)

ndash Denaturing gradient-gel electrophoresis

(DGGE) and related techniques

ndash Carbodiimide modification (CDI)

ndash Chemical cleavage of mismatch (CCM)

ndash Single-strand conformation polymorphism

(SSCP)

ndash Heteroduplex analysis (HET)

ndash Direct sequencing (DS)

Scanning Procedures

RGH Cotton Mutation Research 285 (1993) 125-

144

Screening Procedures

bull Allele-specific oligonucleotide (ASO)

bull Allele-specific amplification (ASA)

bull Ligation (LIG)

bull Primer extension (PEX)

bull Artificial introduction of restriction sites

(AIRS)

Screening Procedures

RGH Cotton Mutation Research 285 (1993) 125-144

Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as

heteroduplexes on hydrolink gels Trends Genet 7 5

bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels

bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity

bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description

bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)

Technological Improvement of HET to eg DHPLC

bull Patent Information1 Column matrix

ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria

ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236

ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA

DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex

Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]

ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]

ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA

ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]

ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]

Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing

ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]

ndash Licensed to Gene Machines Redwood City CA USA

DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific

Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]

Principle of DHPLC

Rapid denaturation of DNA by heating re-annealing by slow cooling

Heteroduplexes form in the presence of two different alleles

DHPLCGraphical Scheme

Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)

Nucleic Acid Amplifications eg PCR

PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System

from Invitrogen

ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture

composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D

polymerase Pyrococcus species GB-D polymerase possesses a proofreading

ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading

enzyme with Taq DNA polymerase at an optimized ratio increases fidelity

approximately eight times over that of Taq DNA polymerase alone and allows

amplification of simple and complex DNA templates The enzyme mixture is

provided with an optimized buffer that improves enzyme fidelity

ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer

formulation have been optimized for use with denaturing high-performance

liquid chromatography (dHPLC) systems They were developed and tested using

the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is

supplied at 1 unit per microl

Equipments

1 Thermocycler (with heated lid)

2 DHPLC WAVE DNA fragment analysis system

(Transgenomic)

3 DNA Sep HT cartridge (cat no DNA-99-3710

Transgenomic)

4 Transmit silicone sealing mats (see Note 2) (cat no

172024 Transgenomic)

5 PCR plates (see Note 3) (cat no 0030 127307

Eppendorf)

6 Chemical waste container

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Scanning Procedures

ndash Ribonuclease cleavage (RNAase)

ndash Denaturing gradient-gel electrophoresis

(DGGE) and related techniques

ndash Carbodiimide modification (CDI)

ndash Chemical cleavage of mismatch (CCM)

ndash Single-strand conformation polymorphism

(SSCP)

ndash Heteroduplex analysis (HET)

ndash Direct sequencing (DS)

Scanning Procedures

RGH Cotton Mutation Research 285 (1993) 125-

144

Screening Procedures

bull Allele-specific oligonucleotide (ASO)

bull Allele-specific amplification (ASA)

bull Ligation (LIG)

bull Primer extension (PEX)

bull Artificial introduction of restriction sites

(AIRS)

Screening Procedures

RGH Cotton Mutation Research 285 (1993) 125-144

Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as

heteroduplexes on hydrolink gels Trends Genet 7 5

bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels

bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity

bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description

bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)

Technological Improvement of HET to eg DHPLC

bull Patent Information1 Column matrix

ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria

ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236

ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA

DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex

Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]

ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]

ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA

ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]

ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]

Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing

ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]

ndash Licensed to Gene Machines Redwood City CA USA

DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific

Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]

Principle of DHPLC

Rapid denaturation of DNA by heating re-annealing by slow cooling

Heteroduplexes form in the presence of two different alleles

DHPLCGraphical Scheme

Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)

Nucleic Acid Amplifications eg PCR

PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System

from Invitrogen

ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture

composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D

polymerase Pyrococcus species GB-D polymerase possesses a proofreading

ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading

enzyme with Taq DNA polymerase at an optimized ratio increases fidelity

approximately eight times over that of Taq DNA polymerase alone and allows

amplification of simple and complex DNA templates The enzyme mixture is

provided with an optimized buffer that improves enzyme fidelity

ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer

formulation have been optimized for use with denaturing high-performance

liquid chromatography (dHPLC) systems They were developed and tested using

the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is

supplied at 1 unit per microl

Equipments

1 Thermocycler (with heated lid)

2 DHPLC WAVE DNA fragment analysis system

(Transgenomic)

3 DNA Sep HT cartridge (cat no DNA-99-3710

Transgenomic)

4 Transmit silicone sealing mats (see Note 2) (cat no

172024 Transgenomic)

5 PCR plates (see Note 3) (cat no 0030 127307

Eppendorf)

6 Chemical waste container

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Scanning Procedures

RGH Cotton Mutation Research 285 (1993) 125-

144

Screening Procedures

bull Allele-specific oligonucleotide (ASO)

bull Allele-specific amplification (ASA)

bull Ligation (LIG)

bull Primer extension (PEX)

bull Artificial introduction of restriction sites

(AIRS)

Screening Procedures

RGH Cotton Mutation Research 285 (1993) 125-144

Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as

heteroduplexes on hydrolink gels Trends Genet 7 5

bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels

bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity

bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description

bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)

Technological Improvement of HET to eg DHPLC

bull Patent Information1 Column matrix

ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria

ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236

ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA

DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex

Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]

ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]

ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA

ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]

ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]

Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing

ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]

ndash Licensed to Gene Machines Redwood City CA USA

DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific

Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]

Principle of DHPLC

Rapid denaturation of DNA by heating re-annealing by slow cooling

Heteroduplexes form in the presence of two different alleles

DHPLCGraphical Scheme

Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)

Nucleic Acid Amplifications eg PCR

PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System

from Invitrogen

ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture

composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D

polymerase Pyrococcus species GB-D polymerase possesses a proofreading

ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading

enzyme with Taq DNA polymerase at an optimized ratio increases fidelity

approximately eight times over that of Taq DNA polymerase alone and allows

amplification of simple and complex DNA templates The enzyme mixture is

provided with an optimized buffer that improves enzyme fidelity

ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer

formulation have been optimized for use with denaturing high-performance

liquid chromatography (dHPLC) systems They were developed and tested using

the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is

supplied at 1 unit per microl

Equipments

1 Thermocycler (with heated lid)

2 DHPLC WAVE DNA fragment analysis system

(Transgenomic)

3 DNA Sep HT cartridge (cat no DNA-99-3710

Transgenomic)

4 Transmit silicone sealing mats (see Note 2) (cat no

172024 Transgenomic)

5 PCR plates (see Note 3) (cat no 0030 127307

Eppendorf)

6 Chemical waste container

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Screening Procedures

bull Allele-specific oligonucleotide (ASO)

bull Allele-specific amplification (ASA)

bull Ligation (LIG)

bull Primer extension (PEX)

bull Artificial introduction of restriction sites

(AIRS)

Screening Procedures

RGH Cotton Mutation Research 285 (1993) 125-144

Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as

heteroduplexes on hydrolink gels Trends Genet 7 5

bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels

bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity

bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description

bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)

Technological Improvement of HET to eg DHPLC

bull Patent Information1 Column matrix

ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria

ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236

ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA

DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex

Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]

ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]

ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA

ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]

ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]

Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing

ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]

ndash Licensed to Gene Machines Redwood City CA USA

DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific

Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]

Principle of DHPLC

Rapid denaturation of DNA by heating re-annealing by slow cooling

Heteroduplexes form in the presence of two different alleles

DHPLCGraphical Scheme

Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)

Nucleic Acid Amplifications eg PCR

PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System

from Invitrogen

ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture

composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D

polymerase Pyrococcus species GB-D polymerase possesses a proofreading

ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading

enzyme with Taq DNA polymerase at an optimized ratio increases fidelity

approximately eight times over that of Taq DNA polymerase alone and allows

amplification of simple and complex DNA templates The enzyme mixture is

provided with an optimized buffer that improves enzyme fidelity

ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer

formulation have been optimized for use with denaturing high-performance

liquid chromatography (dHPLC) systems They were developed and tested using

the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is

supplied at 1 unit per microl

Equipments

1 Thermocycler (with heated lid)

2 DHPLC WAVE DNA fragment analysis system

(Transgenomic)

3 DNA Sep HT cartridge (cat no DNA-99-3710

Transgenomic)

4 Transmit silicone sealing mats (see Note 2) (cat no

172024 Transgenomic)

5 PCR plates (see Note 3) (cat no 0030 127307

Eppendorf)

6 Chemical waste container

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Screening Procedures

RGH Cotton Mutation Research 285 (1993) 125-144

Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as

heteroduplexes on hydrolink gels Trends Genet 7 5

bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels

bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity

bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description

bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)

Technological Improvement of HET to eg DHPLC

bull Patent Information1 Column matrix

ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria

ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236

ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA

DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex

Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]

ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]

ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA

ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]

ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]

Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing

ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]

ndash Licensed to Gene Machines Redwood City CA USA

DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific

Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]

Principle of DHPLC

Rapid denaturation of DNA by heating re-annealing by slow cooling

Heteroduplexes form in the presence of two different alleles

DHPLCGraphical Scheme

Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)

Nucleic Acid Amplifications eg PCR

PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System

from Invitrogen

ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture

composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D

polymerase Pyrococcus species GB-D polymerase possesses a proofreading

ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading

enzyme with Taq DNA polymerase at an optimized ratio increases fidelity

approximately eight times over that of Taq DNA polymerase alone and allows

amplification of simple and complex DNA templates The enzyme mixture is

provided with an optimized buffer that improves enzyme fidelity

ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer

formulation have been optimized for use with denaturing high-performance

liquid chromatography (dHPLC) systems They were developed and tested using

the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is

supplied at 1 unit per microl

Equipments

1 Thermocycler (with heated lid)

2 DHPLC WAVE DNA fragment analysis system

(Transgenomic)

3 DNA Sep HT cartridge (cat no DNA-99-3710

Transgenomic)

4 Transmit silicone sealing mats (see Note 2) (cat no

172024 Transgenomic)

5 PCR plates (see Note 3) (cat no 0030 127307

Eppendorf)

6 Chemical waste container

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Heteroduplex analysis (HET) Keen J et al (1991) Rapid detection of single base mismatches as

heteroduplexes on hydrolink gels Trends Genet 7 5

bull Heteroduplexes containing single base-pair mismatches can be accurately separated from related heteroduplexes on nondenaturing gels

bull Others performed separation of heteroduplexes on normal gels which detected deletions but their method probably would not detect point mutations and can thus be considered a different method of lesser sensitivity

bull Thus far there have been few modifications The main advantage of the HET method is simplicity (as for SSCP) but its application has not been so widespread partly because of its later description and partly because of the need for Hydrolink gels in the initial description

bull The main disadvantage is the lack of 100 detection Like SSCP the HET method can only be applied to fragments hundreds of base pairs long (for example 200-300 bp)

Technological Improvement of HET to eg DHPLC

bull Patent Information1 Column matrix

ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria

ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236

ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA

DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex

Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]

ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]

ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA

ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]

ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]

Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing

ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]

ndash Licensed to Gene Machines Redwood City CA USA

DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific

Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]

Principle of DHPLC

Rapid denaturation of DNA by heating re-annealing by slow cooling

Heteroduplexes form in the presence of two different alleles

DHPLCGraphical Scheme

Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)

Nucleic Acid Amplifications eg PCR

PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System

from Invitrogen

ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture

composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D

polymerase Pyrococcus species GB-D polymerase possesses a proofreading

ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading

enzyme with Taq DNA polymerase at an optimized ratio increases fidelity

approximately eight times over that of Taq DNA polymerase alone and allows

amplification of simple and complex DNA templates The enzyme mixture is

provided with an optimized buffer that improves enzyme fidelity

ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer

formulation have been optimized for use with denaturing high-performance

liquid chromatography (dHPLC) systems They were developed and tested using

the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is

supplied at 1 unit per microl

Equipments

1 Thermocycler (with heated lid)

2 DHPLC WAVE DNA fragment analysis system

(Transgenomic)

3 DNA Sep HT cartridge (cat no DNA-99-3710

Transgenomic)

4 Transmit silicone sealing mats (see Note 2) (cat no

172024 Transgenomic)

5 PCR plates (see Note 3) (cat no 0030 127307

Eppendorf)

6 Chemical waste container

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Technological Improvement of HET to eg DHPLC

bull Patent Information1 Column matrix

ndash 1Bonn G Huber C Oefner P (1994) Verfahren zur Trennung von Nucleinsaeuren Austrian Patent No 398 973 Vienna Austria

ndash 2Bonn G Huber C Oefner P (1996) Nucleic Acid Separation on Alkylated Nonporous Polymer Beads US Patent No 5585236

ndash Currently exclusively licensed to Transgenomic Inc Omaha NE USA

DHPLCndash 3Oefner PJ Underhill PA (1998) Detection of Nucleic Acid Heteroduplex

Molecules by Denaturing High-Performance Liquid Chromatography and Methods for Comparative Sequencing US Patent 5795976 [Stanford Reference] [USPTO]

ndash 4Hansen NF Oefner PJ (1997) Software to Determine Optimum Temperature for DHPLC Given DNA Sequence Stanford University Invention Disclosure S97-175 Tangible Research Property in conjunction with US Patent 5795976 [Stanford Reference]

ndash Currently licensed to Transgenomic Inc Omaha NE USA Agilent Palo Alto CA USA and Varian Walnut Creek CA USA

ndash 5Oefner PJ (1999) Detection of Polymorphisms by Denaturing High-Performance Liquid Chromatography US Patent 6453244 [Stanford Reference] [USPTO]

ndash 6Huber CG OKeefe M Oberacher H Oefner PJ Premstaller A Xiao W Temperature-Modulated Array High-Performance Liquid Chromatography Provisional Patent filed [Stanford Reference]

Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing

ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]

ndash Licensed to Gene Machines Redwood City CA USA

DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific

Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]

Principle of DHPLC

Rapid denaturation of DNA by heating re-annealing by slow cooling

Heteroduplexes form in the presence of two different alleles

DHPLCGraphical Scheme

Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)

Nucleic Acid Amplifications eg PCR

PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System

from Invitrogen

ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture

composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D

polymerase Pyrococcus species GB-D polymerase possesses a proofreading

ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading

enzyme with Taq DNA polymerase at an optimized ratio increases fidelity

approximately eight times over that of Taq DNA polymerase alone and allows

amplification of simple and complex DNA templates The enzyme mixture is

provided with an optimized buffer that improves enzyme fidelity

ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer

formulation have been optimized for use with denaturing high-performance

liquid chromatography (dHPLC) systems They were developed and tested using

the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is

supplied at 1 unit per microl

Equipments

1 Thermocycler (with heated lid)

2 DHPLC WAVE DNA fragment analysis system

(Transgenomic)

3 DNA Sep HT cartridge (cat no DNA-99-3710

Transgenomic)

4 Transmit silicone sealing mats (see Note 2) (cat no

172024 Transgenomic)

5 PCR plates (see Note 3) (cat no 0030 127307

Eppendorf)

6 Chemical waste container

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Technological Improvement of HET to eg DHPLCbull Patent Information2 DNA shearing

ndash 7Oefner PJ Hunicke-Smith S (1998) Apparatus and Methods for Shear Breakage of Poly-nucleotides US Patent 5846832 [Stanford Reference] [USPTO]

ndash Licensed to Gene Machines Redwood City CA USA

DNA markersndash 8Oefner PJ Underhill PA Human Y Chromosome Specific

Single Nucleotide Polymorphisms US Patent Pending [Stanford Reference]

Principle of DHPLC

Rapid denaturation of DNA by heating re-annealing by slow cooling

Heteroduplexes form in the presence of two different alleles

DHPLCGraphical Scheme

Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)

Nucleic Acid Amplifications eg PCR

PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System

from Invitrogen

ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture

composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D

polymerase Pyrococcus species GB-D polymerase possesses a proofreading

ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading

enzyme with Taq DNA polymerase at an optimized ratio increases fidelity

approximately eight times over that of Taq DNA polymerase alone and allows

amplification of simple and complex DNA templates The enzyme mixture is

provided with an optimized buffer that improves enzyme fidelity

ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer

formulation have been optimized for use with denaturing high-performance

liquid chromatography (dHPLC) systems They were developed and tested using

the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is

supplied at 1 unit per microl

Equipments

1 Thermocycler (with heated lid)

2 DHPLC WAVE DNA fragment analysis system

(Transgenomic)

3 DNA Sep HT cartridge (cat no DNA-99-3710

Transgenomic)

4 Transmit silicone sealing mats (see Note 2) (cat no

172024 Transgenomic)

5 PCR plates (see Note 3) (cat no 0030 127307

Eppendorf)

6 Chemical waste container

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Principle of DHPLC

Rapid denaturation of DNA by heating re-annealing by slow cooling

Heteroduplexes form in the presence of two different alleles

DHPLCGraphical Scheme

Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)

Nucleic Acid Amplifications eg PCR

PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System

from Invitrogen

ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture

composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D

polymerase Pyrococcus species GB-D polymerase possesses a proofreading

ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading

enzyme with Taq DNA polymerase at an optimized ratio increases fidelity

approximately eight times over that of Taq DNA polymerase alone and allows

amplification of simple and complex DNA templates The enzyme mixture is

provided with an optimized buffer that improves enzyme fidelity

ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer

formulation have been optimized for use with denaturing high-performance

liquid chromatography (dHPLC) systems They were developed and tested using

the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is

supplied at 1 unit per microl

Equipments

1 Thermocycler (with heated lid)

2 DHPLC WAVE DNA fragment analysis system

(Transgenomic)

3 DNA Sep HT cartridge (cat no DNA-99-3710

Transgenomic)

4 Transmit silicone sealing mats (see Note 2) (cat no

172024 Transgenomic)

5 PCR plates (see Note 3) (cat no 0030 127307

Eppendorf)

6 Chemical waste container

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

DHPLCGraphical Scheme

Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)

Nucleic Acid Amplifications eg PCR

PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System

from Invitrogen

ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture

composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D

polymerase Pyrococcus species GB-D polymerase possesses a proofreading

ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading

enzyme with Taq DNA polymerase at an optimized ratio increases fidelity

approximately eight times over that of Taq DNA polymerase alone and allows

amplification of simple and complex DNA templates The enzyme mixture is

provided with an optimized buffer that improves enzyme fidelity

ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer

formulation have been optimized for use with denaturing high-performance

liquid chromatography (dHPLC) systems They were developed and tested using

the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is

supplied at 1 unit per microl

Equipments

1 Thermocycler (with heated lid)

2 DHPLC WAVE DNA fragment analysis system

(Transgenomic)

3 DNA Sep HT cartridge (cat no DNA-99-3710

Transgenomic)

4 Transmit silicone sealing mats (see Note 2) (cat no

172024 Transgenomic)

5 PCR plates (see Note 3) (cat no 0030 127307

Eppendorf)

6 Chemical waste container

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Software1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) software2 Navigator (Transgenomic)

Nucleic Acid Amplifications eg PCR

PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System

from Invitrogen

ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture

composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D

polymerase Pyrococcus species GB-D polymerase possesses a proofreading

ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading

enzyme with Taq DNA polymerase at an optimized ratio increases fidelity

approximately eight times over that of Taq DNA polymerase alone and allows

amplification of simple and complex DNA templates The enzyme mixture is

provided with an optimized buffer that improves enzyme fidelity

ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer

formulation have been optimized for use with denaturing high-performance

liquid chromatography (dHPLC) systems They were developed and tested using

the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is

supplied at 1 unit per microl

Equipments

1 Thermocycler (with heated lid)

2 DHPLC WAVE DNA fragment analysis system

(Transgenomic)

3 DNA Sep HT cartridge (cat no DNA-99-3710

Transgenomic)

4 Transmit silicone sealing mats (see Note 2) (cat no

172024 Transgenomic)

5 PCR plates (see Note 3) (cat no 0030 127307

Eppendorf)

6 Chemical waste container

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Nucleic Acid Amplifications eg PCR

PCR Amplification for Mutation Analysis Using the Transgenomic WAVEreg System

from Invitrogen

ndash Introduction Discoverasetrade dHPLC DNA Polymerase is an enzyme mixture

composed of recombinant Taq DNA polymerase and Pyrococcus species GB-D

polymerase Pyrococcus species GB-D polymerase possesses a proofreading

ability by virtue of its 3rsquo to 5rsquo exonuclease activity Mixture of the proofreading

enzyme with Taq DNA polymerase at an optimized ratio increases fidelity

approximately eight times over that of Taq DNA polymerase alone and allows

amplification of simple and complex DNA templates The enzyme mixture is

provided with an optimized buffer that improves enzyme fidelity

ndash The Discoverasetrade dHPLC DNA Polymerase enzyme mixture and buffer

formulation have been optimized for use with denaturing high-performance

liquid chromatography (dHPLC) systems They were developed and tested using

the Transgenomic WAVEreg SystemDiscoverasetrade dHPLC DNA Polymerase is

supplied at 1 unit per microl

Equipments

1 Thermocycler (with heated lid)

2 DHPLC WAVE DNA fragment analysis system

(Transgenomic)

3 DNA Sep HT cartridge (cat no DNA-99-3710

Transgenomic)

4 Transmit silicone sealing mats (see Note 2) (cat no

172024 Transgenomic)

5 PCR plates (see Note 3) (cat no 0030 127307

Eppendorf)

6 Chemical waste container

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Equipments

1 Thermocycler (with heated lid)

2 DHPLC WAVE DNA fragment analysis system

(Transgenomic)

3 DNA Sep HT cartridge (cat no DNA-99-3710

Transgenomic)

4 Transmit silicone sealing mats (see Note 2) (cat no

172024 Transgenomic)

5 PCR plates (see Note 3) (cat no 0030 127307

Eppendorf)

6 Chemical waste container

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Solutions1 Optimase polymerase (supplied with Mg2+-free

buffer and separate Mg2+ solution)(cat no 703045 Transgenomic)2 100 mM dNTP set PCR grade (cat no 10297-117

Invitrogen)3 Mutation control standards low-range mutation

standard (cat no 560077) and high-range mutation standard (cat no 562001 Transgenomic)

4 DNA sizing standard (cat no 560078 Transgenomic)5 Acetonitrile 9993+ HPLC grade (cat no 27 071-7

Sigma-Aldrich)6 WAVE ion-pairing agent 2 M triethylammonium

acetate (TEAA) (cat no 553303 Transgenomic)7 Water (eg MilliQ water) use 18 M1048625 resistance or

better8 WAVE optimized buffers (optional) Buffers A B D

and syringe solution (cat nos 553401 553402 553404 and 553403 Transgenomic)

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

HomoduplexesHeteroduplex

es

Chromatographic Separation Reverse-phase ion-pair system

Gradient elution with acetonitrilewater

UV detection

Column oven temperature selected for partial denaturation of heteroduplexes and thus earlier elution

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Results

Example of elution profiles for a wild-type and a mutant sample The mutant sample contains an insertiondeletion mutation in exon 8 of the RET gene

Example of elution profiles obtained for a wild-type and a mutant sample The mutant sample contains a stop codon mutation in exon 15 of the RET gene

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Applications DHPLC Systems (eg WAVE Systems) can

be used for a wide range of applications includingndash Mutation Detectionndash Sizingndash Oligonucleotide Purification and Analysisndash Forensicsndash Microbial Analysisndash RNA Isolation and Purificationndash Single Base Extensionndash Methylation

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

With the WAVE System you can

Scan fragments for both known and novel

mutationsSNPs without extensive resequencing

Detect low-abundance mutations in heterogeneous

samples

Enrich for low-abundance alleles

Fractionate complex mixtures of related fragments

based on differences in sequence content

Perform targeted mutationSNP scoring via primer

extension assays

Analyze end-point products from allele-specific

ProbePrimer-based Amplification Technologies

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

ProbePrimer-based Amplification Technologies

bull Primer-based Amplificationsndash Non-Isothermal Amplifications eg PCR-based

Methodsndash Isothermal AmplificationsNon-PCR-based

Amplificationsndash NASBAndash HDAndash SDAndash and more than 45 NAIA Technologies

bull Probe-based Amplificationsndash For example Ligase Chain Reactions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

CARTRIDGE dependent Applications eg via WAVE Systems

DNA-99-3510 DNASep Cartridge 46 mm x 50 mm for variety of

DNA applications including Sizing and Mutation Detection load

capacity of 2microg

DNA-99-3710 DNASep HT Cartridge 65 mm x 37 mm for variety

of DNA application but especially for Rapid Mutation Detection

load capacity of 4microg

NUC-99-3550 OligoSepreg Cartridge 46 mm x 50 mm for Oligo

Purification work only

NUC-99-3860 OligoSep Prep HC Cartridge 78 mm x 50 mm for

Oligo Purification load capacity of 5000microg

RNA-99-3810 RNASepreg Prep Cartridge 78 mm x 50 mm for

variety of RNA applications load capacity of 50microg total RNA -OR-

1000ng of specific RNA

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Chromatograms and pedigree of patients 3 and 16 A DHPLC analysis in normal control patients 3 and 16 Both patients show abnormal DHPLC peak patterns with two peaks in comparison to normal control B Sequencing data in patient 3 and his family members C Sequencing data in patient 16 and his family members

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Example of Oligo Analysis by DHPLC

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Thanks for Your AttentionsReference

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions

Thanks for Your Attentions

ReferenceMethods in Molecular Medicine Vol 108

Hypertension Methods and Protocols Edited by J P Fennell and A H Baker copy Humana Press Inc Totowa NJ Chapter 13

• DHPLC Principles An Introduction to Denaturing High Performanc
• Nucleic Acids
• Nucleic Acids OMICS
• Genomics Variations as MutationPolymorphism
• The Principles for Gene Variation Analysis
• Scanning Procedures
• Scanning Procedures (2)
• Screening Procedures
• Screening Procedures (2)
• Heteroduplex analysis (HET) Keen J et al (1991) Rapid dete
• Technological Improvement of HET to eg DHPLC
• Technological Improvement of HET to eg DHPLC (2)
• Slide 13
• DHPLC Graphical Scheme
• Software 1 HSM (Hitachi) and WAVEMAKER 41 (Transgenomic) soft
• Slide 16
• Slide 17
• Nucleic Acid Amplifications eg PCR
• Equipments
• Solutions
• Slide 21
• Results
• Applications
• With the WAVE System you can
• ProbePrimer-based Amplification Technologies
• CARTRIDGE dependent Applications eg via WAVE Systems
• Slide 27
• Slide 28
• Slide 29
• Chromatograms and pedigree of patients 3 and 16 A DHPLC analy
• Example of Oligo Analysis by DHPLC
• Slide 32
• Thanks for Your Attentions Reference
• Thanks for Your Attentions