Amplification of DNA using the Polymerase Chain Reaction

(PCR)

Polymerization of nucleotides by DNA polymerase

Phosphodiester bonds are formed by a nucleophilic attack of a free 3’-OH on the 5’--PO4 of the incoming dNTP.

The enzyme DNA polymerase.

A DNA template to guide synthesis.

A primer, a short nucleotide segment complementary to the template that can provide a free 3’-OH for synthesis.

Requirements for DNA replication

Replication by E. coli DNA polymerase III

E. coli DNA polymerase III beta subunit has a doughnut-shaped hole lined with positively-charged amino acid side

chains that interact with the negatively-charged DNA strand

Annual Review of Biochemistry (1995) 64, 176-200. PDB ID 3BEP

DNA polymerase III subunit three-dimensional structure

Thermus aquaticus (Taq) DNA polymerase

DNA polymerase isolated from a bacterium found in hot springs

Can withstand the high temperatures in PCR needed to denature double stranded DNA templates

Can replicate 1000 base pairs of DNA in 10 seconds at 72°C

Has low replication fidelity; error rate of 1 in 9,000 nucleotides

A variety of thermostable polymerases that have a greater fidelity are now available from several companies.

Thermal cycling during PCR

Initial denaturation

Cycle 1 Cycle 2 Cycle 3 Cycle 30

Extension

Annealing

Primer Design for PCROptimal length for PCR is 18–30 nucleotidesPercentage of dG or dC should be between 40% and 60%Avoid complementarity of two or three bases at the 3' ends of primer pairs to reduce primer–dimer formationAvoid mismatches between the primer and the target-template sequence, especially at the 3' end of the primerAvoid a 3'-end dT. Primers with a dT at the 3' end have a greater tolerance of mismatch and may bind to sequences other than the desired sequence. Use a final concentration of 0.1–0.5 μM (pmol/µl) of each primer. Primer concentration of 0.2 μM is usually sufficient.

Primer-Dimers

Formed due to self-priming by one or both primers

Avoid complementary 3’ ends

Each primer-dimer formed serves as a template in the next cycle

Melting temperatureThe melting temperature (Tm) of a primer is defined as the temperature at which half of the oligonucleotide forms a stable double helix with the template DNA and the other half is separated into single stranded species. There are many methods for calculating the melting temperature of a primer-template pair, some very complicated. A simplified formula for estimating melting temperature for an oligonucleotide, up to 20 nucleotides and assuming no mismatched base pairs, is  Tm = 2°C x (A+T) + 4°C x (G+C) Optimal annealing temperatures may be above or below the estimated Tm. As a starting point, use an annealing temperature 5°C below the calculated Tm.

Melting temperatureA more accurate formula that can be used for oligonucleotides between 15 and 70 nucleotides is Tm = 81.5 + 16.6(log(I)) + 0.41(%G+C) – (600/N) Where I is the molar concentration of monovalent cations and N is the length of the oligonucleotide.

Another formula that can be used for oligonucleotides between 20 and 35 nucleotides is

Tm = 22 + 1.46 ((2 x G+C) + (A+T))

DNA polymerases used for PCR

Two classes of DNA polymerase are commonly used according to the template they copy

DNA-dependent DNA polymerasesRNA-dependent DNA polymerases (aka reverse transcriptases)

Two properties are often considered

Processivity –affinity of the enzyme for the templateFidelity – accuracy of synthesis and a measure of the error rate

See http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/PCR/pcr-enzymes-master-mixes.html for an example of choosing a PCR enzyme

Commercial Sources of Taq DNA polymerase

Amplitaq® - Perkin Elmer Applied BiosystemsRecombinant protein produced in E. coliPurified using a heating stepDNA possibly present in the sample may contaminate PCR reactionsAmplitaq® LD (low DNA) or native Taq can be used

Amplitaq Gold®

Taq is chemically modified and inactive until heated at 94ºC for 10 minutes (hot start)Traditional hot start used antibodies for inactivation

Stoffel fragment of TaqLacks 289 N-terminal amino acids of AmplitaqLacks 5’-3’ exonuclease activity of Amplitaq2-fold more thermostable that Amplitaq

Fidelity of Taq DNA polymerase

Taq lacks a 3’-5’ exonuclease “proofreading” activity.Taq will incorporate 1 error in 1000 nucleotidesTaq will cause a frameshift 1 in 4000 nucleotides

Many labs now use thermostable proofreading enzymes that contain 3’-5’ exonuclease activity for PCR reactions

Note: Taq DNA polymerase preferentially adds an adenine to the 3' end of the product. Such PCR amplified inserts can be cloned into linearized vectors that have complementary 3' thymine overhangs.

Properties of some thermostable DNA polymerases

Source: McPherson and Møller. PCR The Basics. Taylor and Francis 2006. Page 38.

Polymerase Mixtures

The use of a mixture containing predominantly Taq with a low concentration of a proofreading enzyme improves fidelity but allows high levels of product to be formed.

Often called “long-range” PCR

Advantage®2 mix from Clontech

Expand™ High Fidelity PCR system from Roche contains Taq and Pwo DNA polymerases

PCR additives or enhancersCertain PCR templates are difficult to amplify, often due to high GC base content. Chemical additives, such as dimethylsulfoxide (DMSO), up to 10%, or formamide, up to 5%, reduce secondary structure and base pairing.

Other additives are:Trimethylammonium chloride (10-100 uM)Betaine (N,N,N-trimethylglycine) 1-1.3MNonionic detergents such as Tween 20 at 0.1-2.5%Polyethylene glycol (PEG) 6000 5-15%Glycerol 10-15%

Many DNA polymerases purchased commercially come with PCR additives or enhancers of unknown identity.

Control reactions and optimization of PCR

Control tubes should be run with:

No DNANo primersSingle primers

The following will need to be optimized:

Magnesium ion concentrationOther ions (At KCl>0.2M DNA denaturation is inhibited)DNA polymerase concentration Temperatures (denaturation, annealing, extension)Cycle number and length (kept to a minimum)Template concentration (may have to dilute contaminants)

Touchdown PCRStarts initially with an annealing temperature higher than the Tm of the primers and then gradually decreases to below the Tm.

This ensures that only specific annealing of the primers to their correct target sequence occurs before any nonspecific annealing events.

Example:Over first 20 cycles start at 65ºC and reduce annealing temperature 1ºC every 2 cycles to 55ºC. Then run 10 more cycles at 55ºC.

Hot start PCRSpecificity problems can arise before the first cycle of PCR. Nonspecific annealing can occur before initial denaturation.Could wait until PCR reaction is at 95ºC before adding DNA polymerase. That is not practical.

The most common hot start is to inactivate DNA polymerase until the 95ºC denaturation temperature is reached. Inhibitory monoclonal antibodies that bind to the polymerase and inhibit are denatured and released at 95ºC.

Wax beads can encapsulate DNA polymerase. The wax melts at high temperature and releases the enzyme. (Taq Bead hot start polymerase from Promega) Magnesium also has been encapsulated in wax beads.

Nested PCR

If PCR produces a mix of desired and undesired products, PCR1 DNA can be used in a second PCR with primers internal to the primer binding sites from the first PCR reaction. The odds of undesired PCR products containing both primer binding sets is essentially zero.

RT-PCRRT-PCR utilizes a reverse transcriptase to produce a complementary DNA (cDNA) product from an RNA template

RT-PCR systems purchased commercially typically contain a reverse transcriptase (like Tth polymerase) and DNA polymerase(s) (like a mixture of Taq and Pwo DNA polymerases)

Reverse transcriptases have two activities:

DNA polymerase activity which requires a RNA or DNA primer is required to initiate synthesis.

RNase H activity: RNase H is a ribonuclease that degrades the RNA from RNA-DNA hybrids, such as are formed during reverse transcription of an RNA template.

RT-PCR, is a useful tool for such things as diagnosing microbial diseases rapidly and a myriad of other applications. In many cases, standard preparations of reverse transcriptase are used for RT-PCR, but mutated forms with relatively high thermal stability have been developed to facilitate the process.

See http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/PCR/reverse-transcription/reverse-transcriptase-enzymes.html for an example.

Standard RT-PCRReverse transcription from mRNA using an oligo-dT primer. Primers can also be specific for a certain gene.

Semi-quantitative RT-PCRDifferences in mRNA levels in samples can be estimated using RT-PCR. The amount of PCR sample produced during the exponential stage of amplification is used for analysis. Primer pairs and reaction conditions must be identical.

Lanes 1&2 – A. thaliana flowersLanes 3&4 – A. thaliana roots

Lane 1 – 10 cycles of PCR Lane 2 – 15 cycles of PCR

Lane 3 – 10 cycles of PCRLane 4 – 15 cycles of PCR

Quantitative RT-PCR

Coamplification of both the target mRNA and a standard mRNA in a single reaction, termed competitor PCR, is used to quantify mRNA levels in a sample. The competitor is designed to have the same primer binding sites.Various known amounts of competitor RNA is mixed with the sample containing the target RNA.

Quantitative real-time PCR (QPCR)

QPCR is used to quantify the amount of a specific DNA present in a sample, often viral or bacterial in origin, or human blood or tissue. The target DNA may be present at high or low levels.

During QPCR, the amount of PCR product formed is measured each cycle and reported in fluorescence units. The more target DNA present in a sample, the more quickly the PCR product (and therefore fluorescence) is generated.

Threshold CycleThe threshold cycle (Ct) value denotes how many cycles of PCR are required for the amount of PCR product (measured by fluorescence) to reach a defined threshold value. The more target DNA present in a sample, the lower the Ct value will be, as the threshold is reached sooner.

Fluorescent detectionDetection of DNA product formed (amplicon) utilizes a molecule such as SYBR green that binds to double-stranded DNA or a TaqMan probe.

The fluorescent dye SYBR Green binds to the minor groove of the DNA double helix. In solution, the unbound dye exhibits very little fluorescence, however, fluorescence is greatly enhanced upon DNA binding.

TaqMan assays exploit the 5'-exonuclease activity of Taq. The probe is labeled with a fluorescent reporter at the 5' end, often fluorescein (FAM), and a quencher dye at the 3' end.

(www.bio-rad.com)

Amplification of DNA using PCRReagents: Taq DNA polymerase

Amplification buffer Magnesium chloride (MgCl2)deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP)Template DNA (E. coli genomic DNA)5’ primer (100 M concentration)3’ primer (100 M concentration)H2O to 50 l volume

Thermocycle parameters: 1. 94ºC for 5 minutes2. 94ºC for 15 seconds3. 45ºC or 55ºC for 30 seconds4. 68ºC for 1 minute5. Go to step 2, repeat 29 times6. 4ºC forever

Plan your PCR reaction