polymerase chain reaction
- Steps in PCR
- Types of PCR
- Polymerase chain reaction is a technique used to amplify the target DNA sequence from the source of DNA, it’s a rapid and a versatile in vitro methods
- This method is introduced by Kary Mullis in 1985.
- This method is able to amplify the specific sequence of the DNA from the heterogeneous collection of DNA molecules. For this selective amplification some prior information of the target DNA sequence is required.
- Two primer synthesises by this sequence information get attached to the target DNA after denaturation (at the specific site). Primer initiates the DNA synthesis in the presence of heat stable DNA polymerase and DNA precursors (dnTPs).
- Technique based on enzymatic replication of DNA
- Short segment of DNA is amplified by primers which are mediated by enzymes.
- DNA polymerase synthesises complementary DNA from the template DNA. DNA polymerase adds nucleotide at 3’-OH group only so to attach DNA polymerase to the strand at specific site primers in required.
- DNA template: segment of the interested DNA from the sample
- DNA polymerase: a heat stable DNA polymerase which can withstand high temperature e.g. Taq polymerase.
- Oiligonucleotide primers: short stretch of DNA complementary to the 3’ ends of sense and antisense strands.
- Deoxyribonucleotide triphosphate: provide energy for polymerization and building blocks for the synthesis of DNA
- Buffer system: provide optimum conditions for DNA denaturation and renaturation, magnesium and potassium are the key component. Important for polymerase activity and stability.
Steps of PCR:
It is a cyclic process because newly synthesized DNA strand serve as the template for further DNA synthesis. It consist three steps:
It is the initial step in which the reaction mixture is heated to the temperature about 90-980 C that ensures the separation of the DNA strands known as DNA denaturation. Duration of denaturation step usually is 2 min at 94oC.
In the steps the mixture is cooled to the temperature that permits the attachment of the primer to the complementary strand of DNA.
Primer attached at the 3’-ends of the two strands.
Duration for annealing is 1 min at 40-600 C.
The temperature is so adjusted that DNA polymerase synthesizes the complementary strand using the primers for attachment at 3’OH end
Primers extended towards each other so that the DNA segment lying in between is copied.
Duration of this step is usually 2 min at 720 C.
Types of PCR:
- Nested PCR
- Quantitative Real-Time PCR
- Inverse PCR
- Anchored PCR
- Touchdown PCR
- Genetic fingerprinting tool
- Identification of criminal’s DNA collected from the crime site among the various people.
- Tests for paternity
- Comparison of two organism’s genome for study
- Gene mapping
- Phylogenetic analysis of DNA from sources like fossils
- Analysis for the expression of genes.
- Detection of the disease causing genes
- Testing of mutation
- Monitoring gene in gene therapy.