Presentation on theme: "Amplification of DNA using the Polymerase Chain Reaction (PCR)"— Presentation transcript:
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’- -PO 4 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, 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.
Primer 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 formation Avoid mismatches between the primer and the target-template sequence, especially at the 3' end of the primer Avoid 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 temperature The melting temperature (T m ) 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 T m = 2°C x (A+T) + 4°C x (G+C) Optimal annealing temperatures may be above or below the estimated T m. As a starting point, use an annealing temperature 5°C below the calculated T m.
Melting temperature A more accurate formula that can be used for oligonucleotides between 15 and 70 nucleotides is T m = (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 is T m = ((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 polymerases RNA-dependent DNA polymerases (aka reverse transcriptases) Two properties are often considered Processivity –affinity of the enzyme for the template Fidelity – accuracy of synthesis and a measure of the error rate See Services/Applications/PCR/pcr-enzymes-master-mixes.html for an example of choosing a PCR enzymehttp://www.invitrogen.com/site/us/en/home/Products-and- Services/Applications/PCR/pcr-enzymes-master-mixes.html
Commercial Sources of Taq DNA polymerase Amplitaq ® - Perkin Elmer Applied Biosystems Recombinant protein produced in E. coli Purified using a heating step DNA possibly present in the sample may contaminate PCR reactions Amplitaq ® 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 Taq Lacks 289 N-terminal amino acids of Amplitaq Lacks 5’-3’ exonuclease activity of Amplitaq 2-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 nucleotides Taq 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 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 enhancers Certain 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 ( uM) Betaine (N,N,N-trimethylglycine) 1-1.3M Nonionic 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.
Control reactions and optimization of PCR Control tubes should be run with: No DNA No primers Single primers The following will need to be optimized: Magnesium ion concentration Other 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 PCR Starts initially with an annealing temperature higher than the T m of the primers and then gradually decreases to below the T m. 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 PCR Specificity 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-PCR RT-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 transcription/reverse-transcriptase-enzymes.html for an example.http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/PCR/reverse- transcription/reverse-transcriptase-enzymes.html
Standard RT-PCR Reverse transcription from mRNA using an oligo-dT primer. Primers can also be specific for a certain gene.
Semi-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 flowers Lanes 3&4 – A. thaliana roots Lane 1 – 10 cycles of PCR Lane 2 – 15 cycles of PCR Lane 3 – 10 cycles of PCR Lane 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 Cycle The 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 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)
Amplification of DNA using PCR Reagents: Taq DNA polymerase Amplification buffer Magnesium chloride (MgCl 2 ) deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP) Template DNA (E. coli genomic DNA) 5’ primer (100 M concentration) 3’ primer (100 M concentration) H 2 O to 50 l volume Thermocycle parameters: 1. 94ºC for 5 minutes 2. 94ºC for 15 seconds 3. 45ºC or 55ºC for 30 seconds 4. 68ºC for 1 minute 5. Go to step 2, repeat 29 times 6. 4ºC forever Plan your PCR reaction