There are various techniques available for DNA analysis, each with some advantages and disadvantages. While DNA discovery has been a significant achievement, the development of new and advanced DNA analysis tools has made major contributions to scientific research and development. PCR is one of the DNA analysis techniques, which is used for in vitro enzymatic amplification of short DNA sequences.
Polymerase Chain Reaction
PCR or the Polymerase Chain Reaction is a technique, which is widely used in various scientific fields including molecular biology, diagnostics, genetics, forensic science, and paternity testing among others. The DNA polymerase is an enzyme, which is used for in vitro DNA replication. The original DNA molecule is replicated by DNA polymerase enzyme and it doubles the number of DNA molecules. A second cycle of replication starts where each of the DNA molecules is replicated again. These cycles are called chain reactions and millions of copies of DNA are produced from a single piece of DNA.
Three major steps are involved in the PCR process: denaturation, annealing (joining), and extension. These steps are repeated for almost 30-40 cycles in an automated cycler. The machine heats and cools the test tubes present in the reaction mixture.
Denaturation at 94****°C: In this process, the double stranded DNA molecule is denatured and opens into single stranded DNA.
Annealing at 54****°C: After denaturation, the single stranded DNA attaches to the primer present in the tube.
Extension at 72 °C: At this temperature, the DNA polymerase enzyme works well and extends the chain further.
PCR technique was developed in the 1983 by Kary Mullis and has become a very useful and sophisticated technique for DNA analysis. Scientists are frequently using this technique in laboratories for DNA cloning, recombinant DNA technology, DNA sequencing, and southern blotting. However, in clinical microbiology, the PCR technique is being used for the diagnosis of microbial infections and epidemiological studies.
There are several PCR techniques available, but conventionally, the PCR technique is used for DNA sequence amplification. However, a modification of the conventional method allows scientists to detect RNA viruses. This somewhat modified technique is known as Reverse transcriptase polymerase chain reaction, or RT-PCR. In this technique, RNA molecule is transcribed into complementary DNA, or cDNA using an enzyme reverse transcriptase and the cDNA is further modified by the PCR technique. In addition, there are other modified PCR techniques such as nested-PCR, multiplex-PCR, quantitative-PCR and real time PCR.