POLYMERASE CHAIN REACTION (PCR)

POLYMERASE CHAIN REACTION (PCR)

1. Polymerase Chain Reaction Gandham . Rajeev Email:[email protected]

2. Polymerase chain reaction (PCR) is technique for generating large quantities of a specified DNA. PCR is a cell free amplification technique for synthesizing multiple identical copies of any DNA of interest. Polymerase Chain Reaction

3. The double-stranded DNA of interest is denatured to separate into 2 individual strands. Each strand is allowed to hybridize with a primer (renaturation). The primer-template duplex is used for DNA synthesis (DNA polymerase). Denaturation , renaturation & synthesis are repeated again & again to generate multiple forms of target DNA. Principle of PCR

4. Requirements for PCR: A target DNA (100-35,000 bp in length). Two primers (synthetic oligonucleotides of 17-30 nucleotides length) that are complementary to regions flanking the target DNA. Four deoxyribonucleotides ( dATP , dCTP , dGTP , dTTP ). Technique of PCR

5. A DNA polymerase that can withstand at a temperature up to 95˚C. PCR involves repeated cycles for amplification of target DNA. Each cycle has 3 stages: Denaturation Renaturation or annealing Synthesis

6. Denaturation : On increasing the temperature to about 95˚C for 1 minute, the DNA gets denatured & two strands separate. Renaturation or annealing: As the temperature of mixture is slowly cooled to about 55˚C, the primers base pair with complementary regions flanking target DNA strands. Stages of PCR

7. Synthesis: The initiation of DNA synthesis occurs at 3'-hydroxyl end of each primer. The primers are extended by joining the bases complementary to DNA strands. The synthetic process is comparable to DNA replication of leading strand. Optimum temperature has to be maintained as required by DNA polymerases.

8. Stages of PCR

9. Taq DNA polymerase, optimum temperature is around 75˚C & E.coli DNA polymerase around 37˚C. The reaction can be stopped by raising the temperature (about 95˚C). Each cycle of PCR takes about 3-5 minutes.

10. PCR & cycles

11. The new DNA strand joined to each primer is beyond the sequence that is complementary to second primer. New strands are referred as long template. They will be used in second cycle. Cycle – 1

12. The DNA strands ( original & newly synthesized long template ) are denatured, annealed with primers & subjected to DNA synthesis. At the end of second round, long templates & short templates (DNA strands with primer sequence at one end & sequence complementary to other end primer) are formed. Cycle – 2

13. The original DNA strands along with long & short templates are starting materials. Denaturation , renaturation & synthesis are repeated. This process is repeated again & again for each cycle. At the end of 32 nd cycle of PCR, about million-fold target DNA is synthesized. Cycle – 3

14. Klenow fragment of E.coli DNA polymerase is used in original technique. This enzyme, gets denatured at higher temperature, therefore, fresh enzyme had to be added for each cycle. Taq DNA polymerase ( Thermus aquaticus ) is heat resistant, not necessary to freshly add this enzyme for each cycle. Sources of DNA polymerases

15. Nested PCR Inverse PCR Anchored PCR Reverse transcription PCR (RT-PCR) Asymmetric PCR Real-time quantitative PCR Random amplified polymorphic DNA (RAPD) Amplified fragment length polymorphism Rapid amplification of cDNA ends (RACE). Variations of PCR

16. Prenatal diagnosis of inherited diseases: PCR is used for prenatal diagnosis of various diseases by using chorionic villus samples or cells from amniocentesis. Sickle-cell anemia, β - thelassema & PKU can be detected. Diagnosis of retroviral diseases: Used for diagnosis of HIV infection. Applications of PCR

17. Diagnosis of bacterial infections: Used for the detection of bacterial infections. E.g. tuberculosis. Diagnosis of cancers: Used for the detection of cervical cancer. PCR in forensic medicine: Used for the identification of criminals.

18. Textbook of Biochemistry – U Satyanarayana References

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