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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

Endpoint PCR Assays in Today’s LabPCR/RT-PCR are Used to Assemble or Detect DNA Sequences

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

MasterPure™ DNA and RNA Purification Kits High Yield Method of Purification for Diverse Samples

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

Reverse Transcription ReagentsReagents for cDNA Synthesis and RT-PCR

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 KASP (Kompetitive Allele Specific PCR) Genotyping Assays

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

Genotyping Transgenic MiceScreening Assays- Mutant or Wild Type?

Her2+/+ Her2+/- WT”: Her2-/-

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 Managerkkleman@lucigen.com

Lucigen Tech Supporttechsupport@lucigen.com(608) 831-90118 am – 5 pm central time

Thank You for Listening-in Today!

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