PCR (polymerase chain reaction) is an in vitro technique used to amplify a specific region of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It involves repeated cycles of heating and cooling of the DNA sample to separate the DNA strands and allow primers to anneal, followed by extension of the primers by a thermostable DNA polymerase. Kary Mullis developed PCR in 1985 and was awarded the Nobel Prize for Chemistry in 1993. Requirements for PCR include a DNA template, primers, Taq polymerase enzyme, dNTPs, buffer solution and magnesium ions. There are several applications and variations of PCR including quantitative real-time PCR, reverse transcription PCR, and inverse PCR.
2. What is PCR?
• PCR targets and amplifies
a specific region of a DNA
strand.
• It is an in-vitro technique
to generate large
quantities of a specified
DNA.
• PCR is ‘photocopier.’
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3. HISTORY
• Kary B. Mullis,
developed PCR in 1985
and was awarded the
Nobel Prize for
Chemistry in 1993.
• PCR machine
=thermocycler
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9. ANNEALING:
• The annealing step allows the hybridisation of
the two oligonucleotide primers, which are
present in excess, to bind to their
complementary sites that flank the target
DNA.
• The annealed oligonucleotides act as primers
for DNA synthesis, since they provide a free 3’
hydroxyl group for DNA polymerase.
• TEMPERATURE :45-60°C
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12. Taq DNA polymerase
• The availability of a thermostable DNA
polymerase enzyme isolated from the
thermophilic bacterium Thermus aquaticus
found in hot springs provided the means to
automate the reaction.
Taq DNA polymerase has a temperature
optimum of 72°C and survives prolonged
exposure to temperatures as high as 96°C and
so is still active after each of the denaturation
steps.
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17. Inverse PCR
Amplification of DNA
of unknown
sequence carried out
from known
sequence.
identification of
sequences flanking
transposable
elements
identification of
genomic inserts
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18. REVERSE TRANSCRIPTION PCR
(RT-PCR)
• for amplifying DNA from RNA.
• Reverse transcriptase reverse
transcribes RNA into cDNA,
which is then amplified by PCR.
• Some thermostable DNA
polymerases used in the PCR
such as Tth have a reverse
transcriptase activity under
certain buffer conditions. 18
19. Quantitative real time PCR(Q-RT PCR)
• It quantitatively measures
starting amounts of DNA,
cDNA or RNA.
• Q-PCR is commonly used to
determine whether a DNA
sequence is present in a
sample and the number of its
copies in the sample.
• QRT-PCR methods use
fluorescent dyes, such
as Sybr Green, EvaGen to
measure the amount of
amplified product in real time. 19
