PCR- POLYMERASE CHAIN REACTION

 PCR- POLYMERASE CHAIN REACTION 

Polymerase chain reaction (PCR) is a technique for rapidly and accurately making multiple copies of a specific segment of DNA. Polymerase chain reaction enables researchers to obtain large amounts of DNA for a variety of experiments and procedures in molecular biology, forensic analysis, evolutionary biology, and medical diagnostics.    

DNA fragments are amplified in the laboratory using the Polymerase Chain Reaction (PCR) method.

Materials:

-          PCR machine

-          PCR tubes/strips

-          Pipettes and tips

-          PCR primer sets (oligonucleotides) specific for each target sequence

-          Taq polymerase enzyme

-          dNTPs (deoxynucleotide triphosphates)

-          DNA template

-          Buffer solution (provided with Taq polymerase)

The general stages for doing a PCR are as follows:

1. Preparation of the reaction mixture: The DNA template, primers, Taq polymerase, buffer, and deoxynucleotide triphosphates (dNTPs) are all components of the PCR reaction mixture. A sterile microcentrifuge tube is filled with the correct quantities of each component to create the reaction mixture.

2. DNA template addition: Include the DNA template in the mixture used for the reaction. The DNA fragment that needs to be amplified is called a DNA template.

3. Primer addition: Forward and reverse primers should be added to the reaction mixture. Taq polymerase uses primers, which are short DNA sequences that anneal to template DNA and act as the catalyst for DNA synthesis.

4. Denaturation: The reaction mixture is heated to 94-95°C for 30 seconds to denature the double-stranded DNA template into single-stranded DNA.

5. Annealing: The temperature is lowered to 50-60°C for 30 seconds to allow the primers to anneal to the template DNA.

6. Extension: The temperature is raised to 72°C for 30 seconds to 2 minutes to allow Taq polymerase to extend the primers and synthesize new DNA strands.

7. Repeated cycles: Steps 4-6 are repeated for 20-40 cycles, depending on the desired amplification level.

8. Final extension: The final extension step is carried out at 72°C for 5-10 minutes to ensure complete extension of all synthesized DNA fragments.

9. Cooling: The reaction is cooled to 4°C to stop the reaction.

After the reaction is complete, the PCR products can be analyzed by gel electrophoresis, DNA sequencing, or other methods. The amplified DNA can be used for a variety of applications, such as cloning, sequencing, and gene expression analysis.

Types of PCR

                                            i.            Multiplex PCR

                                          ii.            Nested PCR

                                        iii.            q-PCR

                                        iv.            Immune capture PCR