Presentation on theme: "Amplification of DNA using the Polymerase Chain Reaction (PCR)"— Presentation transcript:
1Amplification of DNA using the Polymerase Chain Reaction (PCR)
2Polymerization of nucleotides by DNA polymerase Phosphodiester bonds are formed by a nucleophilic attack of a free 3’-OH on the 5’-a-PO4 of the incoming dNTP.
3Requirements for DNA replication 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.
4Replication by E. coli DNA polymerase III FIGURE (part 1) DNA synthesis on the leading and lagging strands. Events at the replication fork are coordinated by a single DNA polymerase III dimer, in an integrated complex with DnaB helicase. This figure shows the replication process already underway (parts (a) through (e) are discussed in the text). The lagging strand is looped so that DNA synthesis proceeds steadily on both the leading and lagging strand templates at the same time. Red arrows indicate the 3′ end of the two new strands and the direction of DNA synthesis. The heavy black arrows show the direction of movement of the parent DNA through the complex. An Okazaki fragment is being synthesized on the lagging strand. The subunit colors and the functions of the clamp-loading complex are explained in Figure
5E. 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
6DNA polymerase III subunit three-dimensional structure Annual Review of Biochemistry (1995) 64, PDB ID 3BEP
7Thermus aquaticus (Taq) DNA polymerase DNA polymerase isolated from a bacterium found in hot springsCan withstand the high temperatures in PCR needed to denature double stranded DNA templatesCan replicate 1000 base pairs of DNA in 10 seconds at 72°CHas low replication fidelity; error rate of 1 in 9,000 nucleotidesA variety of thermostable polymerases that have a greater fidelity are now available from several companies.
9(part 1) Amplification of a DNA segment by the polymerase chain reaction. (a) The PCR procedure has three steps. DNA strands are 1 separated by heating, then 2 annealed to an excess of short synthetic DNA primers (blue) that flank the region to be amplified; 3 new DNA is synthesized by polymerization. The three steps are repeated for 25 or 30 cycles. The thermostable DNA polymerase TaqI (from Thermus aquaticus, a bacterial species that grows in hot springs) is not denatured by the heating steps.
10(part 2) Amplification of a DNA segment by the polymerase chain reaction. (a) The PCR procedure has three steps. DNA strands are 1 separated by heating, then 2 annealed to an excess of short synthetic DNA primers (blue) that flank the region to be amplified; 3 new DNA is synthesized by polymerization. The three steps are repeated for 25 or 30 cycles. The thermostable DNA polymerase TaqI (from Thermus aquaticus, a bacterial species that grows in hot springs) is not denatured by the heating steps.
11(part 3) Amplification of a DNA segment by the polymerase chain reaction. (a) The PCR procedure has three steps. DNA strands are 1 separated by heating, then 2 annealed to an excess of short synthetic DNA primers (blue) that flank the region to be amplified; 3 new DNA is synthesized by polymerization. The three steps are repeated for 25 or 30 cycles. The thermostable Taq DNA polymerase (from Thermus aquaticus, a bacterial species that grows in hot springs) is not denatured by the heating steps.
12Primer Design for PCR Optimal length for PCR is 18–30 nucleotides Percentage 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.
13Primer-Dimers Formed due to self-priming by one or both primers Avoid complementary 3’ endsEach primer-dimer formed serves as a template in the next cycle
14Melting 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, isTm = 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.
15Melting temperatureA more accurate formula that can be used for oligonucleotides between 15 and 70 nucleotides isTm = (log(I)) (%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 isTm = ((2 x G+C) + (A+T))
16DNA polymerases used for PCR Two classes of DNA polymerase are commonly used according to the template they copyDNA-dependent DNA polymerasesRNA-dependent DNA polymerases (aka reverse transcriptases)Two properties are often consideredProcessivity –affinity of the enzyme for the templateFidelity – accuracy of synthesis and a measure of the error rateSee for an example of choosing a PCR enzyme
17Commercial 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 usedAmplitaq Gold®Taq is chemically modified and inactive until heated at 94ºC for 10 minutes (hot start)Traditional hot start used antibodies for inactivationStoffel fragment of TaqLacks 289 N-terminal amino acids of AmplitaqLacks 5’-3’ exonuclease activity of Amplitaq2-fold more thermostable that Amplitaq
18Fidelity 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 nucleotidesMany labs now use thermostable proofreading enzymes that contain 3’-5’ exonuclease activity for PCR reactionsNote: 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.
19Properties of some thermostable DNA polymerases Source: McPherson and Møller. PCR The Basics. Taylor and Francis Page 38.
20Polymerase MixturesThe 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” PCRAdvantage®2 mix from ClontechExpand™ High Fidelity PCR system from Roche contains Taq and Pwo DNA polymerases
21PCR additives or enhancers Certain PCR templates are difficult to amplify, often due to high GC base content. Chemical additives, such asdimethylsulfoxide (DMSO), up to 10%,or formamide, up to 5%, reducesecondary structure and base pairing.Other additives are:Trimethylammonium chloride ( uM)Betaine (N,N,N-trimethylglycine) 1-1.3MNonionic detergents such as Tween 20 at %Polyethylene glycol (PEG) %Glycerol 10-15%Many DNA polymerases purchased commercially come with PCR additives or enhancers of unknown identity.
22Control reactions and optimization of PCR Control tubes should be run with:No DNANo primersSingle primersThe following will need to be optimized:Magnesium ion concentrationOther ions (At KCl>0.2M DNA denaturation is inhibited)DNA polymerase concentrationTemperatures (denaturation, annealing, extension)Cycle number and length (kept to a minimum)Template concentration (may have to dilute contaminants)
23Touchdown 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.
24Hot 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 toinactivate DNA polymerase until the95ºC denaturation temperature isreached. Inhibitory monoclonalantibodies that bind to the polymeraseand 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.
25Nested PCRIf 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.
26RT-PCRRT-PCR utilizes a reverse transcriptase to produce a complementary DNA (cDNA) product from an RNA templateRT-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 for an example.
27Standard RT-PCRReverse transcription from mRNA using an oligo-dT primer. Primers can also be specific for a certain gene.
28Semi-quantitative RT-PCR Differences 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 rootsLane 1 – 10 cycles of PCRLane 2 – 15 cycles of PCRLane 3 – 10 cycles of PCRLane 4 – 15 cycles of PCR
29Quantitative RT-PCRCoamplification 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.
30Quantitative 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.
31Threshold 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.
32Fluorescent detection Detection 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)
33Amplification of DNA using PCR Reagents: Taq DNA polymeraseAmplification bufferMagnesium chloride (MgCl2)deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP)Template DNA (E. coli genomic DNA)5’ primer (100 mM concentration)3’ primer (100 mM concentration)H2O to 50 ml volumeThermocycle 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 foreverPlan your PCR reaction