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PCR - POLYMERASE CHAIN REACTION
Introduction: PCR a 'DNA/RNA photocopier'
PCR uses a few basic everyday molecular biology reagents to make large numbers of copies of a specific DNA in a test tube. While the concept is simple, PCR is a complicated process with many reactants. The concentration of template DNA is initially very low but its concentration increases dramatically as the reaction proceeds and the product molecules become new templates. Other reactants, such as dNTPs and primers, are at concentrations that hardly change during the reaction, while some reactants, such as DNA polymerase, can become limiting. There are significant changes in temperature and pH and therefore dramatic fluctuations in the dynamics of a range of molecular interactions. So, PCR is a very complex process, but one with tremendous power and versatility for DNA analysis.
The polymerase chain reaction (PCR) is a powerful core molecular biology technique. It is an efficient and rapid in vitro method for enzymatic amplification of specific DNA or RNA sequences from nucleic acids of various sources. A simple PCR reaction consists of a set of synthetic oligonucleotide primers that flank the target DNA sequence, target DNA, a thermostable DNA polymerase and dNTPs. A repetitive series of cycles involving template denaturation, primer annealing, followed by extension of the annealed primers, yields tremendous amounts of DNA. Because the strands synthesized in one cycle serve as a template in the next, a million-fold increase in the DNA amount is achieved in just 20 cycles.
PCR was first published in 1985 (Saiki et al., 1985) with Klenow polymerase used as the elongation enzyme. Due to the heat instability of the Klenow polymerase, new enzyme had to be added for every new cycle, and the maximum limit of the product length was 400 bp. In 1988 the first report using DNA polymerase from Thermophilus aquaticus (Taq-polymerase) was published (Saiki et al., 1988). This polymerase greatly enhanced the value of PCR, and the introduction of the automatic programmable heating block in the same report also took the tedious need for three different water baths out of the procedure. Currently the PCR technique is utilized in most molecular biology laboratories as a routine tool which is suitable for performing a great number of different experiments. The method is frequently chosen for conducting experiments, such as cloning, making mutations, sequencing, detecting, typing, etc. (Erlich et al., 1991).
2. The basic reaction
PCR is based on the recognition by a short piece of DNA (the primer) of a sequence on a larger, single stranded fragment of DNA (template strand). When the primer recognizes the template and binds (anneals) to the recognition sequence, the 3'-end of the primer is used by DNA polymerase to synthesize a new DNA strand (elongation). When the temperature is raised, the new DNA strand will melt away (denature) from the template, and the template is once again open for annealing of a new primer when the temperature is decreased. By adding a second primer which recognizes the template strand complementary to the first template, the elongation can proceed in the direction of the first primer. In the first round of elongation, this will ideally double the amount of template strands. In the second temperature cycling, half of the templates for the first primer will be new-synthesized fragments, all terminated where the second primer annealed. When these new fragments are recognized by the first primer, the elongation cannot proceed beyond the second primer, and the synthesized fragments will have a fixed length determined by the distance of the annealing sites of the two primers. New production of template strands take place in every temperature cycle. In this way the DNA sequence between the two primer sequences is amplified exponentially, yielding high concentrations of double-stranded DNA of the same length. The newly-formed double stranded DNA is denatured at 94-97ºC. Primers anneal at 35-72ºC (the exact temperature is primer- and assay dependent), and the new product is synthesized at 72ºC, which is the optimal temperature for the Taq-polymerase.
4. Conclusion
PCR is capable of producing large amounts of DNA fragments from a single piece of template DNA as the amplification increases the amount of fragments produced exponentially. In theory, it is possible to detect a single copy of template DNA by PCR using simple methods. For this reason PCR is used to identify nucleic acid sequences that are only present in very small numbers in the sample to be analyzed.
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