Polymerase chain reaction (PCR) is molecular biology based laboratory technique used for amplification of DNA fragment. DNA is amplified by cycles of heating and cooling with enzyme and substrate. Sufficient copies of DNA can be obtained from desired fragment of DNA.
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Components of PCR
- dsDNA: fragment of DNA which is needed to amplify.
- 2 primers for each strand
- Enzyme: Thermophilus polymerase (Taq polymerase): This enzyme is obtained from Thermophilus aquaticus. It is heat stable DNA polymerase and can withstand heating during process of PCR.
- Deoxyribonucleotides: These are used as substrate for amplification.
- Magnesium: Act as ssential cofactors for DNA polymerase activity.
Process of PCR
- Denaturation: DNA fragment, DNA primers, Taq polymerase and Deoxyribonucleotides are heated to around 95oC. This separates the DNA strands.
- Annealing: Thus heated sample is cooled to around 55oC. This allows DNA primers to anneal to the specific sequence for the amplification on DNA templates.
- Elongation: Temperature is increased to 72oC. At this process, DNA polymerase adds deoxyribonucleotides to the strand. Then replication of the sequences after each primer occurs.
This cycle of heating and cooling is continued till sufficient copies of DNA is obtained.
Real time PCR
Also called as quantitative PCR, it has additional component called SYBR green dye. SYBR green dye gives fluorescence when get bound with dsDNA. This allows monitoring of the amplification process in real time, unlike regular PCR.
Reverse transcription PCR
It is used for amplification of RNA. Reverse transcriptase enzyme is used for formation of complementary DNA. Thus formed complementary DNA is amplified via PCR, real time PCR. Then after sufficient complementary DNA is obtained, this DNA is used for RNA synthesis.
References:
- Harper’s Illustrated Biochemistry Thirty Second Edition.
- Lippincott’s Illustrated Reviews – Biochemistry.
