POLYMERASE CHAIN RACTION

POLYMERASE CHAIN REACTION (PCR):

 Polymerase Chain Reaction (PCR) is a powerful technique for the amplification of specific segments of DNA. The PCR method was developed in the 1980s by Kary Mullis and his colleagues, and it has revolutionized molecular biology and genetics research.

Here are the steps involved in the PCR process:

Step 1: Denaturation

The PCR process begins with the denaturation step, which involves heating the sample to a high temperature (typically 94-98°C) to break apart the hydrogen bonds that hold the two strands of DNA together. This step results in two single-stranded DNA molecules.

Step 2: Annealing

In the annealing step, the temperature is lowered to around 50-65°C to allow the short DNA primers to hybridize or anneal to the complementary single-stranded template DNA sequences. The primers provide a starting point for the polymerase enzyme to extend the DNA strand in the next step.

Step 3: Extension

The extension step is carried out at a temperature of 72°C, which is the optimal temperature for most DNA polymerase enzymes. The polymerase enzyme extends the primers by adding nucleotides to the 3' end of the primers, creating a new DNA strand that is complementary to the template DNA strand. The polymerase enzyme can only add nucleotides in the 5' to 3' direction, so it can only extend the DNA strand in one direction. As a result, the two primers define the region that will be amplified in the PCR reaction.

After the extension step, the cycle is repeated by returning to the denaturation step, allowing the newly synthesized DNA strands to become templates for the next round of PCR amplification. Each cycle of PCR doubles the number of copies of the target DNA sequence, resulting in an exponential increase in the amount of the specific DNA fragment.

"The PCR process typically involves 25-40 cycles of denaturation, annealing, and extension. The number of cycles depends on the amount of starting DNA template and the desired amount of amplification. The amplified DNA fragments can be visualized and analyzed by gel electrophoresis, sequencing, or other molecular techniques".

Applications of PCR :

PCR has many applications in molecular biology and genetics research. One of the most common applications is the detection and quantification of gene expression. In this application, PCR is used to amplify a specific mRNA transcript, which is a copy of the DNA sequence that codes for a particular protein. By quantifying the amount of mRNA transcript in a sample, researchers can determine the level of gene expression.