POLYMERASE CHAIN REACTION (PCR)

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.

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.

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).

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.

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.

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.

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.

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.

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 32nd cycle of PCR, about million-
fold target DNA is synthesized.

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.

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).

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.

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