POLYMERASE CHAIN REACTION (PCR)

• Karry Mullis invented this ingenious method in 1989, & awarded Nobel prize in 1993.
• millions of copies of a particular sequence of DNA can be produced within a few hours.
• PCR  is  a method  of enzymatic  amplification  of a target  sequence  of DNA.
• It is sensitive, selective (specific) and extremely rapidmeans of amplifiing any desired sequence of double stranded DNA.
• DNA  to  be amplified  is  replicated  by DNA polymerase  of  Thermus  aquaticus  (Taq) because  it  is  thermostable.
• Primers  are  amplified  to  produce  desired  sequence  of  DNA.
• Two DNA primers of about 20-30 nucleotides with complementary sequence of the flanking region can be synthesized.

Steps  in  PCR:

• PCR  uses DNA  polyme  rase.
• Each  cycle  doubles  the  amout  of DNA  in  the  sample,  leading  to  exponential  increase.
• Thus amplification after’n’number of cycle in (2)n, twenty cycles provide an amplification of 106 (million) and 30 cycles of 10, (billion).

Step 1: Separation (Denaturation):DNA strands are separated (melted) by heating at 95°C for 15 seconds to 2 minutes

Step 2: Priming (Annealing): The primers are annealed by cooling to 50°C for 0.5 to 2 minutes. The primers hybridise with their complementary single stranded DNA produced in the first step.

Step 3: Polymerization: New DNA strands are synthesized by Taq polymerase.This enzyme is derived from bacteria Thermus acquaticus that are found in hot springs. Therefore the enzyme is not denatured at high temperature. The polymerase reaction is allowed to take place at 72°C for 30 seconds in presence of dNTPs (all four deoxy ribonucleotide triphosphates) and DNA polymerase. 

• As Taq polymerase is not denatured on heating and therefore does not have to be added at each successive cycle.
• Both strands of DNA are now duplicated 
• The steps of 1,2 and 3 are repeated, Thus, 20 cycles provide for 1 million times amplifications. These cycles are generally repeated by auto-mated instrument, called Tempcycler.

Thus following is required in PCR:- 

1. target double standard DNA,
2. two specific primrers
3. a thermostable DNA polymerase
4. Taq polymerase,
5. dNTP

                                                                                                        APPLICATION OF PCR:

Detection of infectious diseases: 

1. AIDS, Tuberculosis, CMV, H1N1, etc.
2. Lyme Disease-joint inflammation from tick bites.
3. Detect 3 sexually transmitted diseases in one swab-herpes, papillomarvirus, chlamydia.
4. PCR can diagnosis even one bacteria or virus present in the specimen.
5. Latent viruses can also be diagnosed.

Detection of Variations and Mutations in Genes

1. Detects people with inherited disorders and carriers
2. Track presence or absence of DNA abnormalities, characteristic to cancer.
3. Prenatal diagnosis of genetic disorders.
4. PCR combined with RE and Southern blotting is used for mutation detection.

PCR and the Law

1. DNA fingerprinting can multiply small amounts of DNA
2. found in blood samples, hair, semen, and other
3. body fluids “contain only very few tubercle bacilli ,cytomegalo virus and HIV

Various types  of PCR:

1. Multipler PCR
2. Reverse Tanscriptase PCR: DNA polymerase derived from thermus thermophillus organism has got additional reverse transcriptase activity and hence is preferred for reverse transcriptase type of PCR.
3. Realtime PCR: a fluorescent dye known as “SYBR green is used to tag the primer, this helps in quantitative detection of PCR material.
4. Invert  PCR
5. Nested PCR.

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