dirty dna is okay - sometimes!
TRANSCRIPT
www.lucigen.com
Dirty DNA is Okay - Sometimes!Obtaining Surefire and Rapid PCR/RT-PCR Results from Diverse Sample Types
Karen Kleman, Ph.D.Product Manager, LucigenJanuary, 2018
AgendaObtaining Surefire and Rapid PCR/RT-PCR Results
• Role of end-point PCR/RT-PCR assays in today’s lab• Challenges in the PCR/RT-PCR workflows• Explore the utility of fool-proof PCR systems for all template
types• Provide examples of quick and effective PCR screening
applications
• Questions
PCR Applications Across IndustriesIdentification or Confirmation of Specific Sequences
Infectious disease detection in Clinical Labs
Authentication of species in the Food Industry
Human identification in forensic sciences
Detection of CRISPR-edited clones in Basic Research
Seed pedigree determination in AgBio
General Workflow for Endpoint PCR/RT-PCR assaysQualitative Analysis of the Sequence of Interest
Harvest Sample Extract DNA or RNA
PCR AmplifyDNA (cDNA)
Analyze Results
Harvest SampleQualitative Analysis of the Sequence of Interest
Harvest Sample Extract DNA or RNA
PCR AmplifyDNA (cDNA)
Analyze Results
Sample Collection and Storage is Important!“Junk In” = “Junk Out”DNA/RNA can degrade or become contaminated during collection & storage
Process fresh samples quickly or add additives (anticoagulants to blood), keep away from UV, heat, etc.
Avoid cross-contamination. Use sterile swabs, self-contained vessels
Inactivate nucleases, or purify before storage
Store properly: 4°C, frozen, or dried
DNA/RNA yields may be low or non-representative Use proper DNA or RNA isolation method Concentrate sample if necessary Amplify (WGA, MessageBOOSTER™ cDNA Synthesis Kit)
Harvest Sample Extract DNA or RNA
Extract DNA or RNA
PCR AmplifyDNA (cDNA)
Analyze ResultsAnalyze Results
Nucleic Acid Extraction from Diverse Samples Sample Type, Quantity, and Amount Dictate the Method
THE GOALS:• Maximize DNA or RNA recovery• Maintain the integrity of the nucleic acid• Remove or inactivate PCR inhibitors and nucleases
Considerations:o What type of sample are you working with?
• Liquid vs solid• Cultured cells vs tissues
o Number of samples• Single or many? High throughput processing and automation
requirements?o Amount of sample & the quantity of the nucleic acid in the sample
• Do you need to enrich or concentrate the DNA or RNA?
Each Sample Type Presents Its Own Unique Challenge(s)Different Sample Types Require Different Extraction Reagents
• Gram positive bacteria have thick cell walls –add Lysozyme
• Yeast and fungi have tough cell walls- may need to grind, add Lyticase or zymolase
• Chitin, collagen, and Keratin are similar-may be able to use the same reagent
• Plants have metabolitessimilar to NA-use young seedlings that contain lower amounts.
• FFPE samples have paraffin and fixatives that must be disrupted or removed
• RBC from mammals do not contain nuclei. Isolate and concentrate WBC
Analyze Results
Inhibitor Sample Type Effect
Polysaccharides Feces, Plant Tissue Affects property of NA, inhibits enzymes,
Polyphenols Plants Affects property of NA, chelates metals
Humic Acids Soil, Plant tissue Binds NA and enzymes
Melanin Hair, Skin Inhibits enzyme
Hematin, Hemoglobin Blood Incomplete melting of DNA, inhibits enzyme
Indigo Denim, Dyes Incomplete melting of DNA, inhibits enzyme
Urea Urine Degradation of enzymes
Immunoglobulin G Blood Inhibits enzyme
Calcium Milk Competes with cofactors of enzyme
DNases, RNases All Degrade nucleic acidhttp://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2012.05384.x/pdf
Beware PCR InhibitorsSample Components Inhibit Enzymes, Alter the Nucleic Acid, Chelate Metals
Strategies to Reduce the Impact of PCR InhibitorsRemoval, Dilution, Denature, or Bind
Analyze Results
Remove inhibitor by precipitation, selective binding, immunocapture
Heat-treat the sample
Add reagents that bind inhibitors
Dilute the nucleic acid sample prior to PCR
Use a robust DNA Polymerase
Isolation of Nucleic Acids From SamplesExtraction is Crude, Purification is Clean (Isolated)
Extraction
Purification
• Sequencing (NGS)• Cloning• Transfection• Expression
“Salting Out” MethodsAn Easy and Fast Method that Uses Non-toxic Reagents
• High salt concentrations to selectively precipitate protein, leaving DNA in solution• DNA is then precipitated out of solution in presence of salt and alcohol
Protein
MasterPure™ DNA and RNA Purification Kits Additions and Spins Only - No Expensive Columns or Beads
Fast: 30-60 minute purification protocol for many sample types
High Purity: A260/A280 ratios consistently between 1.8 and 2.0
High Yields: Improves yields by avoiding the use of columns which often reduce nucleic acid yields
Safe: Uses only non-toxic reagents.
Versatile: Purify TNA, genomic DNA, total RNA, FFPE RNA, or both genomic DNA and total RNA from a sample
Total RNA Recovery: Purify both large and small (e.g., miRNA) RNA for RNA-Seq or qRT-PCR
When Dirty DNA is Not Okay!MasterPure™ DNA and RNA Purification Kits
Kit Time Applications
Masterpure™ Complete DNA and RNA Purification Kit
< 1 hrPurify DNA, RNA, and TNA for PCR, RT-PCR, cloning, sequencing, Northern and Southern blotting, restriction analysis, genomic library preparation
Masterpure™ DNA Purification Kit for Blood Version II
< 40 min Purify genomic DNA from whole blood or buffy coat for PCR, restriction analysis, Southern blotting
Masterpure™ Gram-Positive DNA Purification Kit
30 min -overnight
Purify DNA from Gram-positive bacteria for PCR, fosmid library construction, restriction digestion, Southern blotting
Masterpure™ Yeast DNA Purification Kit < 40 min Purify DNA from yeast for PCR, restriction digestion,
Southern blotting, genomic library preparation
Masterpure™ Yeast RNA Purification Kit < 1 hr Purify RNA from yeast and filamentous fungi for
cDNA synthesis, microarray analysis
Dirty DNA is Okay, Sometimes!Extracted DNA is Abundant and Remains Intact for PCR
Purification
Extraction
End-point PCR screening assays
• Maximize DNA recovery
• Inhibit nucleases, proteases
• Inactivate or diminish PCR inhibitors
QuickExtract™ SolutionsBurst Open the Cells, Heat and Go!
Fast: 8 minute extraction protocol for most sample types
Simple: No centrifugation steps or spin columns, helps increase yields
Automation-friendly: Simple protocol integrates easily into automated workflows
Safe: Uses only non-toxic reagents
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QuickExtract™ DNA and RNA Purification Kits Extraction Solutions for Many Sample Types
Figure 1. FailSafe™ PCR amplifications of genomic DNA extracted from a variety of tissues or cells. Samples were extracted with QuickExtract™ DNA Extraction Solutions. PCR was performed using primers to amplify the regions indicated: Lanes 1-3, human β-globin; lane 4, transgenic mouse GAPDH; lane 5, E. coli 16S ribosomal RNA gene; lane 6, transgenic SV40 T antigen.
Figure 2. Extracted DNA from multiple Zebrafish organs using QuickExtract™ DNA Extraction Solution 1.0. A1-µL aliquot of a 100-µL extracted sample was used to
amplify a single-copy crystallin-like gene. Lane 1, 100-bp ladder; lanes 2-3, fins; lanes 4-5, eyes; lanes 6-7, scales; lane 8, no-DNA control.
Fins
Eyes
Scal
es
Scal
es
Fins
Kit Time Applications
QuickExtract™ DNA Extraction Solution 3-8 min Genotyping, genetic studies, identity testing,
viral/microbial screening, PCR, qPCR
QuickExtract™ RNA Extraction Kit 6 min RT-PCR applications
QuickExtract™ Plant DNA Extraction Solution 8 min PCR, e.g., GMO testing
QuickExtract™ FFPE DNA Extraction Kit 62 min
Microsatellite, single-nucleotide polymorphisms (SNP), tumor heterogeneity studies, copy number variations (CNV), methylation analysis, short tandem repeats (STR)
QuickExtract™ DNA and RNA Purification Kits Extracted Samples Can be Used for PCR, RT-PCR, & qPCR
QuickExtract™ and FailSafe™ PCR SystemHigh Yield RT-PCR Products
Figure 1. Comparative yield of RT-PCR product withdifferent RNA extraction kits. Lysates wereprepared according to manufacturers' instructionsand used as template to produce cDNA using theMMLV RT 1st Strand cDNA Synthesis Kit, followed byPCR using the FailSafe™ PCR System with primersfor the ALDOA gene. Products were separated on a2% agarose gel and stained with SYBR® gold. LaneM, 100 bp ladder; lanes 1 and 2, QuickExtract™ RNAExtraction Kit; lanes 3 and 4, kit from Vendor 1;lanes 5 and 6, kit from Vendor 2.
QE RNA
QE RNA Vendor 1 Vendor 2
ALDOA gene
PCR Amplify DNA
Harvest Sample Extract DNA or RNA
PCR AmplifyDNA (cDNA)
Analyze ResultsAnalyze Results
PCR AmplifyDNA (cDNA)
End-point PCRWhat’s Really Going On in the Reaction Tube?
Considerations:o Template- RNA vs DNA, GC-rich or AT-richo DNA Polymerase: Which type of DNA
polymerase(s) is necessary?o Primers: What’s important when designing
primers?o dNTPs: Does it matter where I source
them?o Buffer (including MgCl2) and Additives-
Optimize for different templates/DNA Polymerases
o Instrumentation and cycling parameters
PCR Template ConsiderationsSequence, Secondary Structure, and Purity
• 1 ng – 1 µg of genomic DNA per PCR reaction, amount may depend on the polymerase
• GC-rich or AT-rich templates may dictate choice of polymerase, buffer conditions.
• May need to remove inhibitors in certain samples
• 10 pg-5 µg of total RNA• Specific primers @ 0.5–1 µM• High throughput• Analysis of 2 genes
• 10 pg-5 µg of total RNA• May use oligo(dT) or random,
primers @ 2-5 µM; or specific primers 0.5–1 µM
• Analysis of multiple genes
Reverse Transcription ReagentsTools for Making cDNA (complementary DNA) from RNA
*May use oligo(dT), random, or specific primers
DNA Polymerases for End-Point PCR Fast, Reliable, and Cost-effective for Screening Applications
• Standard Taq DNA Polymerases Good processivity, fast, cost-effective, but error-prone Best for qualitative end-point assays
• Hot-Start Polymerases Inactivated by bound antibodies or chemicals until heated Used to reduce primer-dimer formation and extension, increase yield. Especially
useful when several primer sets are mixed in one reaction (multiplexing).
• High-Fidelity Polymerases Relatively slower enzyme that has proofreading capabilities Best used for cloning (protein expression) and sequencing
• Polymerases for generating long amplicons Usually a blend of a high processivity enzyme with a proofreading enzyme Buffer adjustments can result in >25kb amplicons
EconoTaq® for Genotyping and Basic PCRReliable “Taq” DNA Polymerase
Data courtesy of Dr. Benjamin Allen, Dept. Molecular & Cellular Biology, Harvard University
Great Taq Polymerase performance at an economical price.Choice of reaction buffers, with or without MgCl2.Non-proofreading PolymeraseSamples available
Characteristics of Good PCR Primers?Think Specificity and Annealing
18-30 bases. For RT-PCR, primers should span exon-exon junctions to avoid amplifying genomic
DNA Contain no internal secondary structure One C or G at 3′ end Have G/C content between 40% and 60% Have a balanced distribution of G/C and A/T rich domains Primer pairs are non-complementary, both internally and to each other
Primer-dimers will form and extend Have a melting temperature (Tm) that allows annealing to occur between 55° and 65°C Primers in a set should have similar melting temperatures. Optimal primer-template annealing temperatures are often approx. 5 to 10°C higher
than the Tm of the primers
Primer-BLAST-https://www.ncbi.nlm.nih.gov/tools/primer-blast/Primer3- https://sourceforge.net/projects/primer3/
Reaction Buffer Can Make All the DifferenceOptimize Buffer for DNA Polymerase and Template Type
dNTPS: Use HPLC-purified stocks, and avoid freeze/thaws. The higher the molarity the faster the reaction (typically 40-200 µM of each).
MgCl2: Acts as a cofactor for the Polymerases. Too high?-reaction can become non-specific. Reduce MgCl2 and dNTPs to amplify target specifically and without interruption.
Salts, pH: The higher the pH, the easier the template DNA disassociates during heating, lower nonspecific binding. High salt keeps the polymerase on the template and stabilizes primer binding.
When the Template is Difficult to AmplifyTry Additives!
Additive Function RecommendedConcentration
Betaine Reduces duplex stability; reduces Tm facilitating amplification of GC-rich regions
0.1 M-3.5 MDo not use Betaine HCL
DMSO Reduces secondary structure, particularlygood for GC-rich templates 2-10%
BSA Binds PCR inhibitors, such as melanin, good when amplifying ancient DNA 0.01-0.10 µg/µL
Triton-X100Tween 20NP-40
Stabilize Taq Polymerase, may reduce secondary structure of template 0.1-1%
Formamide Reduces secondary structure, particularly good for GC-rich templates 1-5%
Reason Solution
Incorrect annealing temperature
Test an annealing temperature gradient, starting at 5°C below the lower Tm of the primer pair
Poor primer design, specificity
Verify that primers do not bind themselves or each otherIncrease length of primerDo they match target sequence
Insufficient primer concentration
Optimize: 0.05–1µM in the reaction
Suboptimal reaction conditions
Optimize MgCl2 0.2-1mM increments
Poor template quality
• Analyze DNA via gel electrophoresis before and after incubation with Mg++
• Check A260/280 ratio of DNA template• Presence of inhibitor in reaction• Decrease sample volume
Insufficient number of cycles Repeat with more cycles
No BAND!
No Band on Your Gel?Try Optimizing the Annealing Temperature and Buffer Conditions
Multiple Bands on Your Gel?Improve Primer Design and Optimize Reaction Conditions
Multiple BANDs!
Reason Solution
Poor primer design
•Increase length of primer•Verify that primers do not bind themselves or each other•Avoid GC-rich 3´ ends
Primer annealing temperature too low Increase annealing temperature
Incorrect Mg++ concentration Adjust Mg++ in 0.2–1 mM increments
Excess primer Primer concentration can range from 0.05–1 µM in the reaction..
Premature replication •Use a hot start polymerase•Set up reactions on ice
Incorrect template concentration
For genomic DNA use 1 ng–1 µg of DNA per 50 µl reaction
Surefire Solutions for PCRFailSafe™ PCR Systems- Avoid the Need to Optimize!
Successful PCR, the first time and every time
PCR amplification of difficult or high-GC templates High Yields:
Multiplex PCR compatible
Robust amplification of targets up to 20 kb long
High accuracy with 3-fold lower error rate than Taq DNA polymerase (error rate = 1 in 30,000)
Extremely high sensitivity and specificity using the PCR Enhancer Technology (with Betaine)
Surefire Solutions for PCR Every TimeTest 12 Pre-Mixes in Parallel and One Will Work
Multiplex !
Amplification of an 80%-85% GC-rich region of the human fragile X gene with the Fail-Safe PCR System.
2X Pre-Mixes
Multiplex PCR amplification of five exons of CFTR from as little as 1 ng of human genomic DNA using the FailSafe System
2X Pre-Mix J worked Best!
When You Know Taq DNA Pol Will Do the TrickEconoTaq® PLUS 2X Master Mixes
Ready-to-use PCR reaction master mixes, contain dNTPs and PCR Enhancer
Can be cycled up to 98°C to amplify the most challenging GC-rich templates others cannot.
EconoTaq PLUS GREEN contains tracking dyes for gel electrophoresis
Outstanding performance and value are perfect for routine PCR
Instrumentation ConsiderationsEach Cycling Step Can be Optimized for Better Results
94–98°C for 1–3 Minutes
• Anneal for 0.5-2 Min• 3–5°C lower than the lowest Tm of the
primers.
Taq DNA Polymerase is 1 min/kb
Cycling ParametersTweaking the PCR Cycling Parameters May Be Required
Higher temps may be required for complex genomic samples & GC-rich templates. Additives? May be able to reduce temp
Polymerases have different extension rates (Taq is fast @ 1 min/kb) and extension time may need to be extended. Annealing & Extension may be combined (2 step)
Remember, when calculating Tm , PCR additives, co-solvents, and modified nucleotides lowers the Tm of the primer-template complex.
Do not want to go much beyond 40 cycles, nonspecific amplification, depletion of reagents. Final extension time should be optimized for amplicon length and complexity.
Analyze Results
Harvest Sample Extract DNA or RNA
PCR AmplifyDNA (cDNA)
Analyze ResultsAnalyze Results
PCR AmplifyDNA (cDNA)
Equipment Considerations for End-Point PCR AnalysisSeparation Based on Size, Fluorescent Readout or Both?
Considerations:o Does your application require a quick answer in the
research lab (screening clones) or a validated method in the clinic (HIV diagnosis)?
o Are you limited to gels, or do you have access to fluorescence-detecting equipment?
o How many samples will be processed at one time?
o Post PCR handling?
End-point PCR Product AnalysisFrom Bands on a Gel to Fluorescent Measurements
Plate readers or qPCR instrumentsGel Electrophoresis
Capillary Electrophoresis
PCR Product AnalysisInstrumentation Considerations
Requirement Gel Capillary PlateReader
qPCRThermocycler
High Throughput -compatible
+ +++ ++++ +++
Multiplex Capabilities
+ ++++ ++ +++
Self-containmentpost PCR
- +++ + ++++
Cost-effective ++++ + ++ ++
Time Savings + ++ +++ ++++
SNP Analysis in AgBio>8000 pre-validated KASP SNP assays for breeding in wheat
KAS genotyping assays for Quantitative Trait Loci (QTL) can be used to survey
potential breed-lines for crop development using these publically
available markers
Identification of CRISPR-Edited Cell Pools or ClonesMany Common Methods Require Target Site Amplification
• PCR amplification of target site followed by Sanger sequencing of amplicons
• Mutations are detected as double peaks, manually or computationally (TIDE)
• SNP detection via qPCR• Can detect loss of wild-type sequence, indicating % editing efficiency
• Indel Detection by Amplicon Analysis, or IDAA™ • Tri-primer amplification followed with analysis by capillary electrophoresis
• Endonuclease Mismatch Cleavage, or EMC Assays (T7E1)• Detect indel mutations (not efficient at detecting single base mutations)
• PCR amplification of target site followed by gel electrophoresis
• Detects large insertions/ deletions
Mutation start point
Modified from German Cancer Research Center (DKFZ), http://www.crisprflydesign.org/direct-sequencing/
Double peaks
Ehrke-Schulz, Mol Ther Methods Clin Dev (2016)3:16047
T7E1 Assay Workflow for Mutation DetectionPoor Quality Sample Prep Yields Poor Assay Results
Analysis by agarose gel
QuickExtract™ is recommended and cited for T7E1 assay sample prep Efficient lysis and extraction Fast protocol Scaleable – 1 sample, or 1000’s Compatible with many high-
fidelity polymerases
Cell lysis and PCR Optimization challenges:• No PCR product• “Smeary” PCR product• Non-specific amplification
No results or unclear assay results
T7EI Assay Results (5 µL PCR product in 20 µL rxn)
PCR Amplification (Q5® Polymerase, 3 µL gDNAin 20 µL rxn, 30 cycles)
Evaluating Gene Editing Efficiency with the T7E1 AssayFast, Scaleable Sample Prep Yields Clear Results
96-well plate of HEK293T cells transfected with Cas9 RNP complex (40K cells/well)
Heteroduplex formation and digestion:• Denaturing and annealing• T7E1 digestion
PCR Amplicon
Parent band
Digest products
Today’s qualitative endpoint PCR Assays are readily used in genotyping applications including, SNP Analysis, transgenic animal analysis, and CRISPR-edited clone identification.
QuickExtract® Extraction Kits provide an easy, simple method for extracting DNA or RNA from a variety of biological samples ranging from bacteria to leaves to human blood cells.
“Quick-extracted” DNA or RNA can be used directly in PCR, RT-PCR, (and qPCR) assays, and is amendable to high throughput extraction workflows.
The FailSafe™ PCR System ensures one-time, surefire success in generating your amplification product.
SummaryReliable End-point PCR Assays can be Readily Performed with Extracted, and not Purified, DNA or RNA
Lucigen Extraction and Purification Kits
Product Cat No.Extraction Kits
QuickExtract™ DNA Extraction Solution QE09050
QuickExtract™ FFPE DNA Extraction Kit QEF81050
QuickExtract™ Plant DNA Extraction Solution QEP70750
QuickExtract™ RNA Extraction Kit QER090150
Purification Kits
MasterPure™ Complete DNA and RNA Purification Kit MC8901
MasterPure™ DNA Purification Kit for Blood Version II MB711400
MasterPure™ Gram Positive DNA Purification Kit MGP04100
MasterPure™ Yeast DNA Purification Kit MPY80200
MasterPure™ Yeast RNA Purification Kit MPY03100
Lucigen PCR Products
Product Cat No.Extraction Kits
EconoTaq® PLUS and EconoTaq PLUS GREEN 2X Master Mixes 30033-1
EconoTaq® DNA Polymerase 30031-1
FailSafe™ PCR Systems FS99060
MessageBOOSTER™ cDNA Synthesis from Cell Lysates Kit MBCL90310
MMLV Reverse Transcriptase 1st-Strand cDNA Synthesis Kit MM070150
Sygnis SunScript™ One Step RT-PCR Kit MPY80200
EpiScript™ RNase H- Reverse Transcriptase ERT12910K
NxGen® M-MuLV Reverse Transcriptase 30222-1
Questions?Please Do Not Hesitate to Contact Me or Tech Support
Contact me.Karen Kleman, Ph.D.Product [email protected]
Lucigen Tech [email protected](608) 831-90118 am – 5 pm central time
Thank You for Listening-in Today!
Extraction and Purification ProductsPCR and Amplfication Products