PCR The Polymerase Chain Reaction

Polymerase Chain Reaction The Polymerase Chain Reaction (PCR) was not a discovery, but rather an invention. A special DNA polymerase (Taq) is used to make multiple identical copies of a short length of DNA (100-10,000 bp) defined by primers. Kary Mullis (mid 1980’s), the inventor of PCR, was awarded the 1993 Nobel Prize in Chemistry.

What is PCR? : Why “Polymerase”? It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase. Why “Chain”? It is called “chain” because the products of the first reaction become substrates of the following one and so on. The “Reaction” Components Target DNA - contains the sequence to be amplified. Pair of Primers - oligonucleotides that define the sequence to be amplified. 3) dNTPs – deoxy-nucleotidetriphosphates: DNA building blocks. 4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction 5) Mg++ ions - cofactor of the enzyme 6) Buffer solution – maintains pH & ionic strength of the reaction solution suitable for the activity of the enzyme

Required a thermostable DNA polymerase Initially PCR used the Klenow fragment of E. coli DNA polymerase - inactivated by high temperatures. Required a thermostable DNA polymerase Taq DNA polymerase from Thermus aquaticus A thermophilic eubacterial µ-org isolated from a hot spring in Yellowstone National Park Kcat = 150 nucleotides/sec/enzyme (at Topt) Taq1/2 = Klenow Fragment (3´→ 5´ exo-) is an N-terminal truncation of DNA Polymerase I which retains polymerase activity but has lost the 5´→ 3´ exonuclease activity Kcat is the "turnover number" and can be calculated as Vmax/[E concentration] and its units are 1/seconds. It basically tells you how much substrate is converted to product in one second. 5 min 97.5 oC 40 min 95.0 oC 130 min 92.5 oC

PCR - before the Thermocycler 8 BORING hours per PCR! 95º C (5 min) 35 times 55º C (3 min) 72º C (5 min)

Development…. PCR work was first published (1985) using Klenow polymerase – unstable with heat New enzyme had to be added manually at each step Maximum length 400bp Great idea – not very practical First reports using DNA polymerase from Thermus aquaticus (1988) Taq-polymerase from Yellow stone National Park hot springs Developed automatic “thermocycler” programmable heat block

Thermocyclers Heated lids Adjustable ramping times Single/Multiple blocks Gradient thermocycler blocks

Standard Tube  Volume  Cost & Evaporation Thin Walled Tube Volume Cost & Evaporation

“Xeroxing” DNA (Xerography, Photocopying) Cycle 1 PLUS dNTPs, buffer, salts,Taq pol, primers Cycle 35 n36 = 68,719,476,736 copies in ~ 2 hrs 2 copies Cycle 2 4 copies Cycle 3 8 copies

A simple Thermocycling Protocol annealing 94ºC 55ºC 72ºC 4ºC 3 min 1 min 45 sec ∞ hold Initial denaturation of DNA 1X 35X extension denaturation

Step 1 Denaturation dsDNA to ssDNA Step 2 Annealing Primers onto template Step 3 Extension dNTPs extend 2nd strand Extension products in one cycle serve as template in the next

Basic Components of PCR Template DNA (0.5 - 50 ng) Oligonucleotide primers (0.1 – 2.0 μM) dNTP’s (20 –250 μM) Thermostable DNA pol (0.5 – 2.5 U/rxn) MgCl2 (1 – 5 mM) affects primer annealing and Taq activity Buffer (usually supplied as 10X) Working concentrations KCL (10 – 50 mM) Tris-HCl (10 mM, pH 8.3) NaCl2 (sometimes) Primers DNA template Buffer + + A C T G MgCl2 dNTPs Taq polymerase

Magnesium ions have a variety of effects MgCl2 (mM) 1.5 2 3 4 5 Magnesium Chloride (MgCl2 - usually 0.5-5.0 mM) Magnesium ions have a variety of effects It acts as cofactor for Taq polymerase Required for Taq to function Mg2+ binds with DNA and affects primer/template interactions Mg2+ influences the ability of Taq pol to interact with primer / template sequences More magnesium leads to less stringency in binding Strength

Taq Pol is active at low temperatures PCR Problems Taq Pol is active at low temperatures At low temperatures mis-priming is likely Temp Extension Rate 55o C 24 nt/sec 37o C 1.5 nt/sec 22o C 0.25 nt/sec 150 nucleotides in 10 min

Primer Design Typically 20 to 30 bases in length Annealing temperature dependent upon primer sequence (~ 50% GC content) Avoid primer complementarity (primer dimer) The last 3 nucleotides at the 3` end is the substrate for DNA polymerase - G or C

Rules for PCR conditions Add an extra 3-5 minute to your cycle profile to ensure everything is denatured prior to starting the PCR reaction Approximate melting temperature (Tm) = [(2 x (A+T)) +(4 x (G+C))]ºC If GC content is < 50% start 5ºC beneath Tm for annealing Temp. If GC content ≥ 50% start at Tm for annealing temperature Extension @ 72ºC: rule of thumb is ~500 nucleotide per minute.

Common PCR additives BSA (Usually at 0.1 - 0.8 µg/µL final concentration) Stabilize Taq polymerase & overcome PCR inhibitors DMSO (Usually at 2-5% v/v, inhibitory at ≤ 10% v/v) Acts as denaturant - good at keeping GC rich template/primer strands from forming secondary structures. Glycerol (Usually at 5-10% v/v) increases PCR efficiency at high temperatures. Stringency enhancers (Formamide, Betaine, TMAC) Concentrations used vary by type Enhances yield and reduces non-specific priming

Typical PCR Temps/Times Initial denaturation 90o – 95o C 1 – 3 min Denature 90o – 95o C 0.5 – 1 min 25 – 40 cycles Primer annealing 45o – 65o C 0.5 – 1 min Primer extension 70o – 75o C 0.5 – 2 min Final extension 70o – 75o C 5 – 10 min Stop reaction 4o C or 10 mM EDTA hold

PROCEDURE …..

PCR Temperature 100 50 T i m e Melting 94 oC 5’ 3’

PCR Temperature 100 50 T i m e Melting 94 oC 5’ 3’ Heat 5’ 3’

PCR T i m e Temperature Melting Melting 94 oC 94 oC Extension 100 50 T i m e Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oC 5’ 3’ 5’ 3’ 5’

PCR T i m e Heat Heat 30x Temperature Melting 94 oC Extension Annealing Primers 50 oC Extension 72 oC Temperature 100 50 T i m e 30x 5’ 3’ Heat 5’ 5’ Heat 5’ 3’ 5’

PCR T i m e 30x Temperature Melting 94 oC Extension Annealing Primers 100 50 T i m e 30x 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 5’

PCR T i m e Heat Heat 30x Temperature Melting 94 oC Extension Annealing Primers 50 oC Extension 72 oC Temperature 100 50 T i m e 30x 3’ 5’ 5’ Heat 5’ 5’ Heat 5’

PCR T i m e 30x Temperature Melting 94 oC Extension Annealing Primers 100 50 T i m e 30x 3’ 5’ 5’ 5’

PCR T i m e 30x Temperature Fragments of defined length Melting 94 oC Annealing Primers 50 oC Extension 72 oC Temperature 100 50 T i m e 30x 3’ 5’ Fragments of defined length 5’ 5’

More Cycles = More DNA Number of cycles 0 10 15 20 25 30 Size Marker

PCR Optimisation 1: Buffers Most buffers have only KCl (50mM) and Tris (10mM) Concentrations of these can be altered KCl facilitates primer binding but concentrations higher than 50mM inhibit Taq DMSO, BSA, gelatin, glycerol, Tween-20, Nonidet P-40, Triton X-100 can be added to aid in the PCR reaction Enhance specificity, but also can be inhibitory Pre-mixed buffers are available

PCR Optimisation 2: MgCl2 MgCl2: required for primer binding MgCl2 affects primer binding, Tm of template DNA, product- and primer-template associations, enzyme activity & fidelity (accuracy). dNTPs, primers and template, chelate the Mg ion, therefore concentration should be higher than dNTPs. Excess magnesium gives non-specific binding. Too little magnesium gives reduced yield.

PCR Optimisation 3: Primer Design Specific to sequence of interest Length 18-30 nucleotides Annealing temperature 50oC-70oC Ideally 58oC-63oC GC content 40-60% 3’ end critical (new strand extends from here) GC clamp (G or C at 3’ terminus) Inner self complementarity: Hairpins <5bp, dimers <9bp

PCR Optimisation 4: Cycling Conditions Denaturation: Some Taq polymerases require initial denaturation (hot start) Annealing temperature: ~ 5oC less than Tm of primers Tm = 4(G + C) + 2(A + T)oC

PCR Optimisation 5: Cycle Number 25-40 cycles Half-life of Taq is 30 minutes at 95oC Therefore if you use more than 30 cycles at denaturation times of 1 min, the Taq will not be very efficient at this point

Types of PCR

Colony PCR Colony PCR- the screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products. Pick a bacterial colony with an autoclaved toothpick, swirl it into 25 μl of Tris-EDTA buffer autoclaved dH2O in an microfuge tube. Heat the mix in a boiling water bath (90-100C) for 2 minutes Spin sample for 2 minutes high speed in centrifuge. Transfer 20 μl of the supernatant into a new microfuge tube Take 1-2 μl of the supernatant as template in a 25 μl PCR standard PCR reaction.

Hot Start PCR This is a technique that reduces non-specific amplification during the initial set up stages of the PCR The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95°C) before adding the polymerase DNA Polymerase- Eubacterial type I DNA polymerase, Pfu These thermophilic DNA polymerases show a very small polymerase activity at room temperature.

Nested PCR Nested PCR is a modification of PCR intended to reduce non-specific binding in products due to the amplification of unexpected primer binding sites. PCR itself is the process used to amplify DNA samples, via a temperature-mediated DNA polymerase. The products can be used for sequencing or analysis, and this process is a key part of many genetics research laboratories, along with uses in DNA fingerprinting for forensics and other human genetic cases. Conventional PCR requires primers complementary to the termini of the target DNA. A commonly occurring problem is primers binding to incorrect regions of the DNA, giving unexpected products. Nested PCR involves two sets of primers, used in two successive runs of PCR, the second set intended to amplify a secondary target within the first run product.

Processes The target DNA undergoes the first run of PCR with the first set of primers. The selection of alternative & similar primer binding sites gives a selection of products, only one containing the intended sequence. The product from the first reaction undergoes a second run with the second set of primers. It is very unlikely that any of the unwanted PCR products contain binding sites for both the new primers, ensuring the product from the second PCR has little contamination from unwanted products of primer dimers, hairpins & alternative primer target sequences.

Multiplex PCR Multiplex PCR is a modification of PCR in order to rapidly detect deletions or duplications in a large gene. This process amplifies genomic DNA samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler. Multiplex-PCR was first described in 1988 as a method to detect deletions in the dystrophin gene. It has also been used with the steroid sulfatase gene. In 2008, multiplex-PCR was used for analysis of microsatellites and SNPs. Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences.

Types of Multiplex PCR Multiplexing reactions can be broadly divided in two categories: Single Template PCR Reaction. This technique uses a single template which can be a genomic DNA along with several pairs of forward and reverse primers to amplify specific regions within a template. 2. Multiple Template PCR Reaction. It uses multiple templates and several primer sets in the same reaction tube. Presence of multiple primers may lead to cross hybridization with each other and the possibility of mis-priming with other templates.

Primer Design Parameters for Multiplex PCR Design of specific primer sets is essential for a successful multiplex reaction. The important primer design considerations are; Primer Length Multiplex PCR assays involve designing of large number of primers, hence it is required that the designed primer should be of appropriate length. Usually, primers of short length, in the range of 18-22 bases are used. 2. Melting Temperature Primers with similar Tm, preferably between 55°C - 60°C are used. For sequences with high GC content, primers with a higher Tm (preferably 75°C-80°C) are recommended. A Tm variation of between 3°-5° C is acceptable for primers used in a pool. 3. Specificity It is important to consider the specificity of designed primers to the target sequences, while preparing a multiplex assay, especially since competition exists when multiple target sequences are in a single reaction vessel..

4. Avoid Primer Dimer Formation The designed primers should be checked for formation of primer dimers, with all the primers present in the reaction mixture. Dimerization leads to unspecific amplification. All other parameters are similar to standard PCR primer design guidelines Applications of Multiplex PCR Pathogen Identification High Throughput SNP Genotyping Mutation Analysis Gene Deletion Analysis Template Quantitation Linkage Analysis RNA Detection Forensic Studies

Reverse Transcriptase PCR Based on the process of reverse transcription, which reverse transcribes RNA into DNA and was initially isolated from retroviruses. First step of RT-PCR - "first strand reaction“-Synthesis of cDNA using oligo dT primers (37°C) 1 hr. “Second strand reaction“-Digestion of cDNA:RNA hybrid (RNaseH)- Allows the detection of even rare or low copy mRNA sequences by amplifying its complementary DNA.

Applications of PCR Classification of organisms Genotyping Molecular archaeology Mutagenesis Mutation detection Sequencing Cancer research Detection of pathogens DNA fingerprinting Drug discovery Genetic matching Genetic engineering Pre-natal diagnosis