PCR stand for Polymerase Chain Reaction :Requirements, Steps involved and Application
The technique was developed by Kary Mullis in 1985 and received Nobel prize for chemistry in 1993.
PCR is an invitro DNA replication method known as polymearse chain reaction in which multiple copies of the gene(DNA) of interest is synthesized.
PCR is so powerful technique that from single copy of DNA molecule, millions of copies can be obtained with high accuracy, specificity and in very short time.
- Double stranded DNA which serve as template strand
- Two sets of primers (small chemically synthesized oligonucleotides that are complementary to the regions of DNA)
- Enzyme: A thermostable DNA polymerase, taq polymerase (isolated from a bacterium, Thermus aquaticus) and the vent polymerase (from Thermococcus litoralis)
- Mg2+ required for Taq enzyme activity.
- Tris Buffer
PCR Steps involved
The three steps of the reaction are 1) denaturation 2) annealing of primers and 3)primer extension.
In principle PCR involves three temperature cycle:
- Denaturation of double stranded DNA is carried out by applying high temperature of 950C for 15 seconds. Each separated single stranded acts as a template for DNA synthesis.
- Annealing of primers: It is carried out in two sets of primers (Forward and Reverse primers), which are added and anneal to the 3'end of each separated strand.Temperature 40-600C anneals two complementary primers to ends of separated single strands of target DNA. Primers act as initiator of replication.
- Primer extension Is done by DNA polymerase (Taq polymerase or vent polymerase) is used in the reaction, which can tolerate the high temperature (720C)of the reaction.
These steps are repeated many times to (30 cycles) obtain several copies of desired DNA.
Polymerase Chain Reaction (PCR) Steps, Requirements and Applications
Gene Cloning vs PCR
Kary Mullis Autobography
Applications of PCR
- Diagnosis of genetic diseases.
- DNA fingerprinting: Forensic medicine
- for molecular mapping
- Study polymorphism
- For preparation of molecular markers.
- Diagnosis of diseases by detection of virus or bacteria.
- Gene therapy and genetic engineering experiments