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POLYMERASE CHAIN REACTION (PCR)
1.
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 95หC.
๏ 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 95หC
for 1 minute, the DNA gets denatured & two
strands separate.
๏ Renaturation or annealing:
๏ As the temperature of mixture is slowly
cooled to about 55หC, 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.
8.
9. ๏ Taq DNA polymerase, optimum
temperature is around 75หC & E.coli DNA
polymerase around 37หC.
๏ The reaction can be stopped by raising the
temperature (about 95หC).
๏ Each cycle of PCR takes about 3-5 minutes.
10.
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, ฮฒ-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.