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POLYMERASE CHAIN REACTION (PCR)

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POLYMERASE CHAIN REACTION (PCR)

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POLYMERASE CHAIN REACTION (PCR)

  1. 1.  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.
  2. 2.  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.
  3. 3.  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).
  4. 4.  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
  5. 5.  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.
  6. 6.  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.
  7. 7.  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.
  8. 8.  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.
  9. 9.  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.
  10. 10.  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 32nd cycle of PCR, about million- fold target DNA is synthesized.
  11. 11.  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.
  12. 12.  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).
  13. 13.  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.
  14. 14.  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.
  15. 15.  Textbook of Biochemistry – U Satyanarayana

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