rapid cycle pcr, real time analysis, and hi-res melting
TRANSCRIPT
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Rapid Cycle PCR, Real Time Analysis, and Hi-Res Melting
Carl Wittwer
Department of PathologyUniversity of Utah
ARUP
Idaho Technology
AMP, Oct. 31, 2008
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Impatient, Lazy, and Cheap
• Rapid Cycle PCR– Fast
• Real-Time Analysis– High information content
• High-Resolution Melting– Low cost
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Cycles
• Detection• Quantification
0 302010
• Detection• Quantification
Cycles
• Detection• Quantification
0 302010
Amplification
Time (min)0 5 10 15
Temperature (°C)
• Product ID• Allele ID
908070
20
Melting Analysis
Temperature (°C)
• Product ID• Allele ID
908070
Amplification & Analysis
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Rapid Cycle PCR
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Temperature cycling for PCR
95°C1 min
55°C2 min
72°C3 min
30 cycles
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Hair Dryer
Re-circulating
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Academic Prototypes
Wood (1988)
Tin (1989)
Plastic (1990)
Electronics
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Better Temperature Control
Amplification of a 536 bp β-globin fragment
from human genomic DNATime (min)
Sam
ple
Tem
pera
ture
(°C
)GelAnalysis
Time for 30Cycles (hr)Temperature Profiles
536 bp
Anal Biochem 1990;186:328-31, Biotechniques. 1991;10:76-83
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Late 1990: The Girlfriend Series
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Company PrototypesCompany Products
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Sample Containers
Flame-Sealed Capillary Tubes
The LightCycler Capillary Cuvette
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Other Containers for Rapid PCR
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Microfluidics and Rapid Cycling
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35 Cycles in <5 min(modifed LightScanner 32)
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Real Time Analysis
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Ethidium Bromide / Transilluminator
Monitoring PCR with Fluorescence
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1993: Monitoring Fluorescence during Amplification
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1994: RapidCycler + Fluorimeter
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1995: LightCycler Prototype
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1996: LightCycler 24
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Licensing to Boehringer Mannheim (1997)
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1998: Roche LightCycler
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1998 – Instrument Comparision
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LightCycler 480
LightCycler 2.0•Adoption by Clinical Laboratories
•First FDA Approved Genetic Tests (2003)
•Open Platform
1998-2008: LightCycler 10-year Anniversary
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Cycle Number
Fluo
resc
ence
Real-Time Monitoring of PCR
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Real-Time Parameters
1. Time
2. Temperature
3. Fluorescence• Once Each Cycle (Quantification)
Probe melting
• With Changing Temperature
Product melting
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Temperature vs. Time(Rapid Cycle PCR)
SYBR Green I Hybridization Probes
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Fluorescence vs. Time(Real Time PCR)
SYBR Green I Hybridization Probes
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Temperature (°C)
SYBR Green I Hybridization Probes
Fluo
resc
ence Product Tm
Probe Tm
Fluorescence vs. Temperature(Hybridization)
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Closed-Tube GenotypingPopular Probe Designs
Two Additions Three Additions
***One probe needed for each allele***
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Real Time SNP Genotyping
Hydrolysis Probes(TaqMan®)
***One probe required for each allele***
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Dual Hybridization Probes(HybProbes®)
Am J Pathol. 1998;153:1055-61
Genotyping by Melting
***Two probes identify many alleles***
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Single Hybridization Probe(SimpleProbe®)
Anal Biochem. 2001;290:89-97
Genotyping by Melting
***One probe identifies many alleles***
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Unlabeled Probe(Luna Probe®)
Clin Chem. 2004;50:1328-35
Genotyping by Melting
***One probe identifies many alleles***
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Genotyping by Melting
***One probe identifies many alleles***
Snapback Primer
Clin Chem. 2008;54:1648-56
Probe Element
(5’-tail)Primer
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Temperature (°C)
-dF/
dTDual Hybridization Probes
(Factor V Leiden)
HeterozygousHomozygous MutantHomozygous WT
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Single Hybridization Probes(CFTR exon 10)
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Unlabeled Probe(Factor V Leiden)
Clin Chem 2005; 51: 1770 - 1777
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Relative Gene Dosage Assessment in SMA
Unlabeled Probe
Amplicon
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Unlabeled Probes for HSV Detection
Clin Chem 53: 1847-1854, 2007
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Snapback Genotyping (SNPs)
G/TC/G
C/TA/C
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Snapback Primers(length of probe element)
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High-Resolution Melting
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Temperature (°C)
Fluo
resc
ence
β -globin 53.2 536Prostate 60.3 292Specific Antigen
Amplification %GC Length bpHepatitis B 50.0 180
Melting Curves for Different Products (SYBR Green I)
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Heterozygote Amplification
TwoHomoduplexes
TwoHeteroduplexes
Observed Combinationof 4 Duplexes
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Heteroduplex Detection with dsDNA Dyes
Patrinos GP and Ansorge WJ, eds, Molecular Diagnostics, 2nd ed, ElsevierChapter 18, in press, 2009
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HR-1Idaho Technology
LightCycler® 480RocheLightScanner®
Idaho Technology
High Resolution Melting Instruments
High Resolution (>=16 bit):• Temperature• Fluorescence
High Data Density (>=10/°C)Rapid Melting (0.1-0.3°C/sec)
Rotor-Gene® 6500HRMCorbett
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Instrument Comparison
Clin Chem 52:494-503;2006, 53:150-2;2007, 53:1544-8;2007.
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Beta-Globin Genotyping/Scanning(110 bp)
Hb C
G A
Hb S
A T
Hb A = wild type
(Clin Chem 2003; 10:853-860)
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Homo
Hetero
Melting Curve (Original Data)
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Melting Curve (Normalized)
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Curve Overlay (high temperature region is matched)
Heterozygotes as a group are distinguished from the homozygotes
Hetero
Homo
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Small Amplicon Genotyping
(Human Platelet Antigens - J Mol Diagn 8:97-104;2006)
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0
20
40
100
80
60
Fluo
resc
ence
(Nor
mal
ized
)
Temperature (C)82 858483
CC
TT
TC SNP typing on a544 bp amplicon. (SNP is in the low temperature domain)
Single Base Genotyping in Long Amplicons
Clin Chem 49:853-60;2003.
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Coagulation Quadraplex
Het
MUT WT
Het
MUT
WT
Het
MUTWT
HetMUT
WT
-dF/
dT
Temperature (°C)
Factor V MTHFR 1298 MTHFR 677 Prothrombin
Clin Chem 54:108-15;2008.
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Scanning for Heterozygotes
• All use PCR first
Specimen Amplify DNA
High-Resolution Melting
Load on matrix
Electrophoresis
dHPLC
Clean upEnzymatic reactionsClean up
Mass Spec
Arrays
Sequencing
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X-linked Chronic Granulomatous Disease
CYBB13 exons
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Missense Mutation
pro to his
Chronic Granulomatous DiseaseFamily Study
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Hi-Res Melting forGenetic Disease Scanning
• Published studies (19)– BRCA1/2, CFTR, F8, NF2, ApoB…
– Sensitivity 99.3% (n=839)– Specificity 98.8% (n=2,659)
Patrinos GP and Ansorge WJ, eds, Molecular Diagnostics, 2nd ed, ElsevierChapter 18, in press, 2009
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Scanning Summary• Inexpensive (PCR reagents + dye)• Rapid (PCR time + melting)• Detects “all” heterozygous variants • Most heterozygous variants have unique melting
curves (93%)• Detects minor allele fractions better than
sequencing• Does not detect large insertions or deletions• Requires good PCR
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Hi-Res Melting of HLA
1 2
3 4 5 6 7 8 9 10
1415
11 16 17
1312
Match instead of genotype
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Exon 2 Exon 3
HLA-B
HLA-C
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Variety Identification of Grapevines
John Mackay et al., Linnaeus Laboratory, Plant Methods 2008, 4:8.
Dinucleotide Repeats (bp)• Pinot (238, 244)• Syrah (244, 250)• Sauvignon Blanc (244, 246)• Chardonnay (242, 244)
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Summary• PCR can be fast• You don’t need labeled probes for homogenous
genotyping– Unlabeled Probes– Snapback Primers
• High Resolution Melting– Small amplicon genotyping– Heterozygote Scanning – Sequence Matching
• HLA• Repeated sequences
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Thanks!Idaho TechnologyNIHARUPRoche Applied ScienceUniversity of Utah
http://dna.utah.edu