POLYMERASE CHAIN REACTION (PCR)
Polymerase Chain Reaction technique was devised by Kary Mullis in 1980s. Before designing this technique, DNA amplification can be done only by cloning method which was time-consuming. The PCR is used to produce enormous numbers of copies of specified DNA sequence without resorting to cloning.
The Versality of the PCR is enormous and the combination of the PCR and sequencing is an extraordinarily powerful tool for the genetic analysis. The most spectacular demonstration of this is the molecular identification of the Magnolia plant for the Miocene Period which is about 18 million years old which is possible only because of PCR. It is used for cloning genes, for invitro mutagenesis, in mapping and sequencing large genomes, and in diagnosing human inherited disorders. In 1985 there were but three research reports on the PCR. 5 years later it is being used in thousands of laboratories. The PCR has indeed revolutionized the practice of molecular genetics.
PCR utilizes Replication principle. It might be called as Invitro replication. DNA Polymerase, Primers and trinucleotides used in PCR. Single stranded DNA molecules for DNA polymerase provided by denaturation process. Initiation of polymerization possible with the primer, therefore the starting point for DNA synthesis can be specified by supplying an oligonucleotide primer that anneals to the template. Both strands can serve as templates for synthesis provided an oligonucleotide primer is supplied for each strand. After primer annealed to template, DNA synthesis continued up to the end. The reaction mixture is then heated to separate the old and newly synthesized strands, which are then available for further cycles. In each cycle new primers annealed to templates because primers are extended during the polymerization unlike in replication. The amplified number of copies DNA molecules after "n" cycles obtained from the formulae "2n".
The starting material for a PCR is DNA that contains the sequence to be amplified. It is not necessary to isolate the sequence to be amplified, because it is defined by the primers used. The oligonucleotide primers directing the starting points for DNA synthesis, DNA polymerase and a mixture of all four deoxynucleotide precursors are added to a tube containing the DNA. The total volume is usually 100 microlitre. The next step in the process is to heat the reaction mixture at about 94*C for 5 minutes. At this temperature, the double stranded DNA molecules separate completely, forming single strands that become the templates for the primers and DNA polymerase. The temperature is then lowered to allow the oligonucleotide primers to anneal to the complementary sequences in the DNA molecules. This annealing temperature is a key variable in determining the specificity of a PCR, so temperatures and times used vary depending on the sequences to be amplified. This generates the primed templates for DNA polymerase.
For the next step, the temperature is raised to 72*C , the optimal temperature for the heat-stable Taq DNA polymerase. The temperature is held at 72*C for up to 5 minutes for DNA synthesis to proceed. At the end of this period, the temperature is raised once more to 94*C , but now for only 20 seconds, so that the short stretches of double stranded DNA to separate. These single strands become templates for another round of DNA synthesis, and the cycle of heating to separate strands, annealing of primers and DNA synthesis by polymerase is repeated for as many as 30 to 60 cycles. At the end of 30 cycles, around 268,435,456 copies of DNA molecules obtained in PCR.
Initially, in PCR E.Coli DNA polymerase is used. This is added manually for each cycle. It become tedious. This defect overcome by adding Taq DNA polymerase from Thermus aquaticus which lives in water at a temperature of 75*C. This enzyme is reasonable stable even at 94*C. The another disadvantage of PCR is the error rate it is one incorrect nucleotide for about every 2 X 104 nucleotides incorporated. This problem can be reduced by beginning the PCR with a large number of template molecules.
1. PCR is used to isolate and amplify Human-Specific DNA sequences. For example Alu PCR is used for this purpose.
2. PCR is used to amplify and produce even single stranded DNA molecules which can be directly utilized for sequencing process. Example is asymmetric PCR which produce single stranded DNA
3. PCR is utilized for the detection of Mutations. For example ras mutations studied using PCR.
4. PCR amplification is used for monitoring cancer therapy which is important to stop chemotherapy.
5. PCR amplification can also be used for the diagnosis of bacterial and viral infections. For example HIV and TB diagnosed with PCR.
6. PCR amplification is used for sex determination of Prenatal Cells.
7. PCR methods utilized to carry out linkage analysis using sing sperm cells.
8. PCR techniques are used in studies of Molecular evolution