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PCR

 Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) is used to copy sequences of DNA in vitro, mimicking the DNA replication. As in replication, a DNA polymerase enzyme is needed to catalyze the reaction of elongation of the new DNA strand. The raw materials needed are the deoxyribonucleotide triphosphate (dNTPs), which are the four deoxyribonucleotides (dATP, dGTP, dTTP and dCTP). A single stranded template DNA is required. Since the DNA polymerase cannot initiate DNA replication, a primer is also needed. In PCR, a primer is a specific sequence of DNA with a small number of nucleotides (oligonucleotide) which is complementary to a sequence at 3’ end of the target DNA to be copied. To copy both the strands two primers, each binding to either of the two strands at 3’ end, are used. In the cell, the primer is an RNA sequence. In addition to these, Mg2+ is also required. These become the ingredients in PCR mix.

The DNA fragment in which the sequence to be copied is present in dsDNA, and hence it needs to be denatured. Denaturation is done by heating the PCR mix to 95 oC. At this temperature most of the enzymes will denature, and therefore it may be necessary to add DNA polymerase after the denaturation. However, the enzymes of the thermophilic organisms are resistant to high temperature. Therefore, the common heat resistant DNA polymerase used in PCR is Taq DNA polymerase which is obtained from thermophilic bacterium Thermus aquaticus. The primer will bind to the complementary sequence of the denatured template DNA. This happens at a lower temperature and this step is called annealing. The annealing temperature will depend on the sequence and the length of the primer. Once the annealing of the primer is achieved, then the primer extension (DNA synthesis) will take place at a different temperature. This is the optimum temperature of the DNA polymerase used. When sufficient time is given, a complementary copy of the template DNA is completed. At the end of this first thermal cycle (denaturation, annealing and extension temperatures), one copy of each strand is produced. However, these are longer than the intended copy of the target DNA sequence (Figure 7.39). After a couple of PCR cycle, an exact copy of the target DNA is synthesized. After this, copies of the target DNA will be produced in an exponential manner (i.e. 2, 4, 8, 16, etc.) after each cycle. A typical PCR will have 35 to 40 cycles. At the end, millions of copies of the desired DNA sequence will be produced from a single template DNA molecule.

 




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