Polymerase Chain Reaction

Polymerase chain reaction

HISTORY

Kary Banks Mullis, Ph.D. (born December 28, 1944) is an American biochemist and was awarded the Nobel Prize in Chemistry in 1993 for his development of the Polymerase Chain Reaction (PCR).

•Spiegelman and Edward Hall discovered the hydrogen bonding between complementary sequences of DNA & RNA. •Arthur Kornberg has identified the first DNA polymerase and He was awarded Nobel Prize in 1959. •In 1976 a DNA polymerase is isolated from T. aquaticus. It is found to retain its activity at temperatures above 75°C. •In 1977 Frederick Sanger reports a method for determining the sequence of DNA. -- The technique involves an oligonucleotide primer, DNA polymerase and modified nucleotide precursors that block further extension of the primer in sequence-dependent manner. He is awarded the Nobel Prize in 1980.

PCR The Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying relatively large quantities of DNA The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that is used by the bacterium Thermus aquaticus that was discovered in hot springs The primary materials, or reagents, used in PCR are: - DNA nucleotides, the building blocks for the new DNA - Template DNA, the DNA sequence that you want to amplify - Primers, single-stranded DNAs between 20 and 50 nucleotides long (oligonucleotides) that are complementary to a short region on either side of the template DNA - DNA polymerase, a heat stable enzyme that drives, or catalyzes, the synthesis of new DNA

PCR The cycling reactions : There are three major steps in a PCR, which are repeated for 20 to 40 cycles. This is done on an automated Thermo Cycler, which can heat and cool the reaction tubes in a very short time. Denaturation at around 94°C : During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example the extension from a previous cycle). Annealing at around 54°C : Hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. If the primers exactly fit the template, the hydrogen bonds are so strong that the primer stays attached Extension at around 72°C : The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)

PCR The different steps of PCR

PCR

Exponential increase of the number of copies during PCR

PCR

Every cycle results in a doubling of the number of strands DNA present After the first few cycles, most of the product DNA strands made are the same length as the distance between the primers The result is a dramatic amplification of a the DNA that exists between the primers. The amount of amplification is 2 raised to the n power; n represents the number of cycles that are performed. After 20 cycles, this would give approximately 1 million fold amplification. After 40 cycles the amplification would be 1 x 1012

Verification of PCR product

PCR and Contamination

The most important consideration in PCR is contamination Even the smallest contamination with DNA could affect amplification For example, if a technician in a crime lab set up a test reaction (with blood from the crime scene) after setting up a positive control reaction (with blood from the suspect) cross contamination between the samples could result in an erroneous incrimination, even if the technician changed pipette tips between samples. A few blood cells could volitilize in the pipette, stick to the plastic of the pipette, and then get ejected into the test sample Modern labs take account of this fact and devote tremendous effort to avoiding cross-contamination

Optimizing PCR protocols

PCR can be very tricky

While PCR is a very powerful technique, often enough it is not possible to achieve optimum results without optimizing the protocol Critical PCR parameters: - Concentration of DNA template, nucleotides, divalent cations (especially Mg2+ ) and polymerase - Error rate of the polymerase (Taq, Vent exo, Pfu) - Primer design

Primer design

Primer design in PCR Perhaps the most critical parameter for successful PCR is the design of primers

Primer selection Critical variables are: - primer length - melting temperature (Tm) - specificity - complementary primer sequences - G/C content - 3’-end sequence Primer length

- primer length is proportional to annealing efficiency: in general, longer the primer, the more inefficient the annealing

- the primers should not be too short as specificity decreases

the

Primer design

G/C content - ideally a primer should have a near random mix of nucleotides, a 50% GC content - there should be no PolyG or PolyC stretches that can promote non-specific annealing

Primer design Melting temperature (Tm) - the goal should be to design a primer with an annealing temperature of at least 50°C - the relationship between annealing temperature and melting temperature is one of the “Black Boxes” of PCR - a general rule-of-thumb is to use an annealing temperature that is 5°C lower than the melting temperature - the melting temperatures of oligos are most accurately calculated using nearest neighbor thermodynamic calculations with the formula: Tm = H [S+ R ln (c/4)] –273.15 °C + 16.6 log

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[K+]

(H is the enthalpy, S is the entropy for helix formation, R is the molar gas constant and c is the concentration of primer) - a good working approximation of this value can be calculated using the Wallace formula: Tm = 4x (#C+#G) + 2x (#A+#T) °C - both of the primers should be designed such that they have similar melting temperatures. If primers are mismatched in terms of Tm, amplification will be less efficient or may not work: the primer with the higher Tm will mis-prime at lower temperatures; the primer with the lower Tm may not work at higher temperatures.

Taq polymerase •Taq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated

•T. aquaticus is a bacterium that lives in hot springs and hydrothermal vents, and Taq polymerase was identified as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR. Therefore it replaced the DNA polymerase from E.coli originally used in PCR. Taq's temperature optimum for activity is 7580°C, with a half-life of 9 minutes at 97.5°C, and can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72°C

One of Taq's drawbacks -is its relatively low replication fidelity. It lacks a 3' to 5' exonuclease proofreading activity, and has an error rate measured at about 1 in 9,000 nucleotides. Some thermostable DNA polymerases have been isolated from other thermophilic bacteria and archaea, such as Pfu DNA polymerase, possessing a proofreading activity, and are being used instead of (or in combination with) Taq for high-fidelity amplification.

Other Enzymes •Pfu DNA polymerase is an enzyme from hyperthermophilic archaeon Pyrococcus furiosus. Pfu is used to quickly amplify DNA in polymerase chain reaction (PCR) processes. 'proofreading' properties compared to other thermostable polymerases. Pfu DNA polymerase possesses 3' to 5' exonuclease proofreading activity. •Vent enzyme from thermococcus litoralis.

Application of PCR • Isolation of genomic DNA PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as generating hybridization probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region. • DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing. Bacterial colonies (E.coli) can be rapidly screened by PCR for correct DNA vector constructs. • Genetic fingerprinting; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.

•Amplification and quantitation of DNA Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample – a technique often applied to quantitatively determine levels of gene expression. Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.

•PCR in diagnosis of diseases PCR permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.

•VNTR PCR involves few modifications to the basic PCR process, but instead targets areas of the genome that exhibit length variation. The analysis of the genotypes of the sample usually involves simple sizing of the amplification products by gel electrophoresis. Analysis of smaller VNTR segments known as Short Tandem Repeats (or STRs) is the basis for DNA Fingerprinting databases such as CODIS.

Hot-start PCR is a technique that modifies the way that a PCR mixture is initially heated. During this step the polymerase is active, but the target has not yet been denatured and the primers may be able to bind to non-specific locations (or even to each other). The technique can be performed manually by heating the reaction components to the melting temperature (e.g. 95°C) before adding the polymerase. Alternatively, specialized systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody, or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. 'Hotstart/cold-finish PCR' is achieved with new hybrid polymerases that are inactive at ambient temperature and are only activated at elevated temperatures.

In Colony PCR bacterial colonies are rapidly screened by PCR for correct DNA vector constructs. Colonies are sampled with a sterile toothpick and dabbed into a master mix. To free the DNA for amplification, PCR is either started with an extended time at 95°C (when standard polymerase is used), or with a shortened denaturation step at 100°C and special chimeric DNA polymerase. Colonies from the master mix that shows the desired product are then tested individually.

Pretreatments and extensions The basic PCR process can sometimes precede or follow another technique: RT-PCR (or Reverse Transcription PCR) is a common method used to amplify, isolate, or identify a known sequence from a cell's or tissue's RNA. PCR is preceded by a reaction using reverse transcriptase, an enzyme that converts RNA into cDNA. The two reactions are compatible enough that they can be run in the same mixture tube, with the initial heating step of PCR being used to inactivate the transcriptase. Also, the polymerase described below exhibits RT activity, and can carry out the entire combined reaction. RT-PCR is widely used in expression profiling, which determines the expression of a gene or identifies the sequence of an RNA transcript (including transcription start and termination sites and, if the genomic DNA sequence of a gene is known, to map the location of exons and introns in the gene). The 5' end of a gene (corresponding to the transcription start site) is typically identified by an RT-PCR method named RACE-PCR, short for Rapid Amplification of cDNA Ends. (Note that the acronym RT-PCR has more recently been applied to Real-Time PCR, a version of Quantitative PCR described above.)

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