Polymerase chain reaction 2018

1. Polymerase Chain Reaction (PCR)
This is an in-vitro method for amplification of a certain sequence of DNA.

2. The reaction requires the following:
a- Two oligonucleotide primers.
b- Thermostable DNA polymerase (Taq polymerase).
c- All the four deoxyribonucleoside triphosphates.

3. PCR is done in cycles. Each cycle comprises the following three steps:
1- Denaturation: The mixture is heated to 95oC for 30 sec. to denature
the DNA and separate the two strands.
2- Primer annealing: The mixture is cooled to ~50 oC to allow the two
primers to bind to the two strands of the target DNA.
3- Elongation: The mixture is heated to 72 oC to allow the polymerase to
elongate each primer by copying the single stranded template.
4- Cycles are repeated: In each cycle the target DNA is doubled. After 10
cycles the DNA is multiplied 103 times. After 30 cycles the DNA is
doubled 109 times.

4. Applications of PCR:
1- It allows synthesis of DNA in sufficient quantities for sequencing or
cloning.
2- It helps in studying the molecular basis of genetic diseases and aids
their prenatal diagnosis e.g. sickle cell anemia.
3- Detection of low-abundance nucleic acid sequences e.g. viruses that
have long latency period as in case of HIV.
4- The use of PCR, recombinant DNA technology and cloning make
possible the synthesis of large quantities of human proteins (e.g. insulin),
synthesis of vaccines (e.g.hepatitis B virus) and synthesis of antibodies
(e.g. monoclonal antibodies).
5- Forensic analysis of DNA samples.
6- Gene therapy for certain genetic diseases.