Polymerase Chain Reaction

Prepared by Ralph John O. Ugalino Arnelson Arwin G. Atis Chemistry 102.2

http://www.xxpresspcr.com/category/pcr-background/

Purpose
PCR is an in vitro method of nucleic acid synthesis by which a particular DNA segment can be specifically replicated
sequencing mutagenesis genetic analyses diagnosis of inherited disorders forensic analyses tracking evolution identifying individuals

GENE AMPLIFICATION

White, T.J. Preface. PCR Protocols: A Guide to Methods and Applications. (1990). xvii-xviiii

DENATURATION

Basic principle

primers

PRIMER ANNEALING

PRIMER EXTENSION

Voet, D., Voet, J. Biochemistry, 4th Ed. (2011). 115 http://www.mun.ca/biology/scarr/PCR_simplified.html

Sample preparation

TaqMastermix + primers + template in nuclease-free water TaqMastermix

Tris-HCl buffer, 10 mM pH 8.6

standard buffer for DNA

dNTPs, 0.2 mM each

Taq DNA polymerase

prevent misincorporation
thermostable

MgCl2, 1.5 mM

joins to nucleotides to be recognized by polymerase; affects annealing, strand dissociation, product specificity

Innis, M.A., Gelfand, D.H. Optimization of PCRs. PCR Protocols: A Guide to Methods and Applications. (1990). 3-12 Saiki, 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.

Sample preparation
Glycerol (gelatin), 5%

TaqMastermix + primers + template in nuclease-free water

TaqMastermix
enzyme stabilizer stimulates nucleic acid synthesis

Tween20, 0.05%
KCl, 50 mM

isolate only from few cells (< 104) to minimize cellular debris heme-degradation products and EDTA may inhibit PCR
Innis, M.A., Gelfand, D.H. Optimization of PCRs. PCR Protocols: A Guide to Methods and Applications. (1990). 3-12 Saiki, 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.

Profile: Taq Polymerase

withstands repeated exposure to T ~ 95C from Thermus aquaticus, a thermophile optimum T: 70-75C processivity: > 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). 129-141

Primers
LCO primer HCO primer 5' GGT CAA CAA ATC ATA AAG ATA TTG G 3 5' TAA ACT TCA GGG TGA CCA AAA AAT CA 3'

universal PCR primers for COI gene COI: 710 bp, mitochondrial cytochrome c oxidase subunit I high degree of sequence conservation at 3 ends across 15 taxa one primer for each strand Annealing T = 50C
Folmer, O., et al. Molecular Marine Biology and Biotechnology. (1994). 3. 294-299.

Thermocycling program
T, C t, s Purpose

2 3 4 5 6

120 ensure total strand separation Repeat steps 2 to 4 @ 41 cycles 94 30 denaturation 55 30 primer annealing 72 60 primer extension 72 300 further elongation 4 delay close strands

94

http://montreal-biotech.com/Products/?link=TPersonal+Thermocycler

Innis, M.A., Gelfand, D.H. Optimization of PCRs. PCR Protocols: A Guide to Methods and Applications. (1990). 3-12 Saiki, 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.

http://www.mun.ca/biology/scarr/PCR_simplified.html

PCR cycling in detail

SCREENING PHASE
OBJECTIVE: selective primer binding to target criticality of primer design Hot-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 detail

EXPONENTIAL AMPLIFICATION PHASE
Great excess of amplified target sequence over genomic template easier scanning 2N products given N cycles limited 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 detail

PLATEAU PHASE
PCR product accumulates, reagents consumed slows PCR down self-annealing of strands primers cannot bind! excess of free primers more likely bind to spurious targets nonspecific replicates and primerdimers OPTIMIZE NUMBER OF CYCLES!

Kidd, K.K., Ruano, G. Optimizing PCR. PCR 2: A Practical Approach. (1995). 1-22.

Principles of primer design

primer sequence unique for region to be amplified no self-homology random base composition low melting temperature of amplified region between primers annealing sites spaced at 100-600 bp

Sharrocks, A.D. The design of primers for PCR. PCR Technology: Current Innovation. (1994). 5-10.

TECHNIQUE: Multiplex PCR

employ 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.

TECHNIQUE: 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.

TECHNIQUE: Semiquantitative PCR

use internal DNA standard and densitometry

Kang J, et al. Quantification of DNA and RNA by PCR. PCR 2: A Practical Approach. (1995). 119-133.

TECHNIQUE: 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

TECHNIQUE: Asymmetric PCR

for DNA sequencing which require single strands use unequal concentration of sense and antisense strand primers

1,4,7: primer A > B 2,5,8: primer B > A 3,6,9: primer A = B

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

Nobel Prize in Chemistry 1993

for his invention of the polymerase chain reaction (PCR) method

http://www.nobelprize.org/nobel_prizes/chemistry/la ureates/1993/mullis-facts.html

Kary B. Mullis

His invention is highly original and significant, virtually dividing biology into the two epochs before PCR and after PCR. New York Times

http://www.nytimes.com/1998/09/15/science/scientist-at-work-kary-mullis-after-the-eureka-a-nobelist-drops-out.html?pagewanted=all&src=pm

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