Slide 1

PolymeraseChain ReactionPrepared by

Ralph John O. UgalinoArnelson Arwin G. AtisChemistry 102.2

http://www.xxpresspcr.com/category/pcr-background/PurposePCR is an in vitro method of nucleic acid synthesis by which a particular DNA segment can be specifically replicatedgene amplification sequencing mutagenesis genetic analyses diagnosis of inherited disorders forensic analyses tracking evolution identifying individualsWhite, T.J. Preface. PCR Protocols: A Guide to Methods and Applications. (1990). xvii-xviiiiBasic principle

DENATURATIONPRIMER ANNEALINGPRIMER EXTENSIONprimers

Voet, D., Voet, J. Biochemistry, 4th Ed. (2011). 115http://www.mun.ca/biology/scarr/PCR_simplified.htmlSample preparationTaqMastermix + primers + templatein nuclease-free waterTaqMastermixTris-HCl buffer, 10 mM pH 8.6standard buffer for DNAdNTPs, 0.2 mM eachprevent misincorporationTaq DNA polymerasethermostableMgCl2, 1.5 mMjoins to nucleotides to be recognized by polymerase; affects annealing, strand dissociation, product specificityInnis, M.A., Gelfand, D.H. Optimization of PCRs. PCR Protocols: A Guide to Methods and Applications. (1990). 3-12Saiki, R.K. Amplification of genomic DNA. PCR Protocols: A Guide to Methods and Applications. (1990). 13-20.Kidd, K.K., Ruano, G. Optimizing PCR. PCR 2: A Practical Approach. (1995). 1-22.TaqMastermix 2x from New England Biolabs, Inc.(2) pH changes with T: 7.8-6.84Sample preparationTaqMastermix + primers + templatein nuclease-free waterTaqMastermixGlycerol (gelatin), 5%enzyme stabilizerTween20, 0.05%KCl, 50 mMstimulates nucleic acid synthesisInnis, M.A., Gelfand, D.H. Optimization of PCRs. PCR Protocols: A Guide to Methods and Applications. (1990). 3-12Saiki, R.K. Amplification of genomic DNA. PCR Protocols: A Guide to Methods and Applications. (1990). 13-20.Kidd, K.K., Ruano, G. Optimizing PCR. PCR 2: A Practical Approach. (1995). 1-22. isolate only from few cells (< 104) to minimize cellular debris heme-degradation products and EDTA may inhibit PCRTaqMastermix 2x from New England Biolabs, Inc.(2) pH changes with T: 7.8-6.8(3) OPTIMIZATION

5Profile: Taq Polymerasewithstands repeated exposure to T ~ 95Cfrom Thermus aquaticus, a thermophileoptimum T: 70-75Cprocessivity: > 60 bases/s @ 70C 2 kb length in just 1 min!very sensitive to low-frequency targets

Gelfand, D.H., White, T.J. Thermostable DNA polymerases. PCR Protocols: A Guide to Methods and Applications. (1990). 129-141The extraordinary ability of Taq DNA polymerase-mediated PCR to amplify rare targets will facilitate the detection of low-frequency events catalyzed by the polymerase6Primersuniversal PCR primers for COI geneCOI: 710 bp, mitochondrial cytochrome c oxidase subunit Ihigh degree of sequence conservation at 3 ends across 15 taxaone primer for each strandAnnealing T = 50C

LCO primer5' GGT CAA CAA ATC ATA AAG ATA TTG G 3

HCO primer5' TAA ACT TCA GGG TGA CCA AAA AAT CA 3'Folmer, O., et al. Molecular Marine Biology and Biotechnology. (1994). 3. 294-299.L = light; H = heavyLCO = forward; HCO = reverse7Thermocycling program

http://www.mun.ca/biology/scarr/PCR_simplified.htmlhttp://montreal-biotech.com/Products/?link=TPersonal+ThermocyclerT, Ct, sPurpose194120ensure total strand separationRepeat steps 2 to 4 @ 41 cycles29430denaturation35530primer annealing47260primer extension572300further elongation64delayclose strandsInnis, M.A., Gelfand, D.H. Optimization of PCRs. PCR Protocols: A Guide to Methods and Applications. (1990). 3-12Saiki, R.K. Amplification of genomic DNA. PCR Protocols: A Guide to Methods and Applications. (1990). 13-20.Kidd, K.K., Ruano, G. Optimizing PCR. PCR 2: A Practical Approach. (1995). 1-22.Top always at T = 104 C prevent condensation!8PCR cycling in detailSCREENING PHASEOBJECTIVE: selective primer binding to targetcriticality of primer designHot-start PCR: higher annealing T Booster PCR: lower primer concentration reduce frequency of mispriming!

Kidd, K.K., Ruano, G. Optimizing PCR. PCR 2: A Practical Approach. (1995). 1-22.PCR cycling in detailEXPONENTIAL AMPLIFICATION PHASEGreat excess of amplified target sequence over genomic template easier scanning2N products given N cycleslimited by sufficiency of primers, enzymes and nucleotides

Kidd, K.K., Ruano, G. Optimizing PCR. PCR 2: A Practical Approach. (1995). 1-22.PCR cycling in detailPLATEAU PHASEPCR product accumulates, reagents consumed slows PCR downself-annealing of strands primers cannot bind!excess of free primers more likely bind to spurious targets nonspecific replicates and primer-dimersOPTIMIZE NUMBER OF CYCLES!

Kidd, K.K., Ruano, G. Optimizing PCR. PCR 2: A Practical Approach. (1995). 1-22.thermodynamic driving force: molar excess of reagents11Principles of primer designprimer sequence unique for region to be amplifiedno self-homologyrandom base compositionlow melting temperature of amplified region between primersannealing sites spaced at 100-600 bp

Sharrocks, A.D. The design of primers for PCR. PCR Technology: Current Innovation. (1994). 5-10.3 G/C clamp due to strong H bond primer-dimers, secondary structures12Technique: Multiplex PCRemploy different primers for multiple target sequences

Hernandez-Rodriguez, P., Ramirez, A.G. Polymerase chain reaction: types, utilities and limitations. Polymerase Chain Reaction. (2012). 157-172.

3 G/C clamp due to strong H bond primer-dimers, secondary structures13Technique: Reverse transcription PCR (RT-PCR)study mRNA by reverse transcription into cDNA which is subsequently amplified

Edwards, J., et al. cDNA cloning by RT-PCR. PCR 2: A Practical Approach. (1995). 89-118.Frohman, M.A. RACE: Rapid amplification of cDNA ends. PCR Protocols: A Guide to Methods and Applications. (1990). 28-38.

Difficult reverse transcription!14Technique: Semiquantitative PCRuse internal DNA standard and densitometry

Kang J, et al. Quantification of DNA and RNA by PCR. PCR 2: A Practical Approach. (1995). 119-133.3 G/C clamp due to strong H bond primer-dimers, secondary structures15Technique: Real-time/quantitative PCR (qPCR)employ fluorescence detection systems such as intercalating agents (Sybr Green) or labeled probes with fluorophores

Hernandez-Rodriguez, P., Ramirez, A.G. Polymerase chain reaction: types, utilities and limitations. Polymerase Chain Reaction. (2012). 157-172.http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechQPCR.shtml

5 nuclease activity of Taq to degrade probe!16Technique: Asymmetric PCRfor DNA sequencing which require single strandsuse unequal concentration of sense and antisense strand primers

McCabe, P.C.. Production of single-stranded DNA by asymmetric PCR. PCR Protocols: A Guide to Methods and Applications. (1990). 76-83.

1,4,7: primer A > B

2,5,8: primer B > A

3,6,9: primer A = BDouble-strand closes immediately cannot sequence!17

Kary B. MullisNobel Prize in Chemistry 1993for his invention of the polymerasechain reaction (PCR) methodhttp://www.nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis-facts.htmlHis invention is highly original and significant, virtually dividing biology into the two epochs before PCR and after PCR. New York Timeshttp://www.nytimes.com/1998/09/15/science/scientist-at-work-kary-mullis-after-the-eureka-a-nobelist-drops-out.html?pagewanted=all&src=pm(1) PCR first unveiled at American Society of Human Genetics Conference, October 1985181243561871243568712435687