Success criteria - PCR

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Success criteria - PCR

• By the end of this lesson we will be know
• The polymerase chain reaction (PCR) is a
  technique for the amplification ( making many
  millions of copies of the original ) of DNA in
  vitro i.e. in the lab.
• In PCR, primers are complementary to specific
  target sequences at the two ends of the region to
  be amplified.
• The stages of PCR involve
• DNA is heated to separate the strands.
• Cooling allows primers to bind to target
  sequences.
• Heat tolerant DNA polymerase then replicates the
  region of DNA.
• Repeated cycles of heating and cooling amplify
  this region of DNA.

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Producing copies by PCR
In order to sequence DNA or carry out DNA
fingerprinting it is necessary to produce a
huge number of exact copies of the original
stands. The technique used to do this is known
as PCR or Polymerase Chain Reaction. Once the
copies of DNA have been produced they can be
analysed. Note This is the technique used by
forensics to amplify tiny samples of DNA for
fingerprinting
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The Polymerase Chain Reaction (PCR)
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DNA strands unzip
Primers attach
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Taq
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Taq
Taq polymerase
Taq
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Understanding - PCR
1.PCR can amplify any DNA sequence hundreds of
millions of times in just a few hours. It is
especially useful because it is highly specific,
easily automated and capable of amplifying minute
amounts of sample 2.The whole process is only
possible because of a special heat-stable enzyme
called Taq polymerase, isolated from thermophilic
bacteria. 3.The enzyme Tac polymerase is able to
tolerate temperatures of 95?C and has a
temperature optimum of 72?C. 4.This enzyme can
synthesise the complementary strand of a given
DNA strand in a mixture containing the four DNA
nucleotide bases and two short DNA fragments
called primers. Each primer is usually about 20
base pairs (bp) long. The primers are designed
to bind to the DNA at either side of the target
sequence.
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• Procedure
• Step 1 The DNA is heated to 950 C breaking the
  hydrogen bonds and separating the strands.
• Step 2 The strands are cooled to between 55
  700C and the primers added.
• Step 3 The strands are heated to between 70
  72 0C so that Taq Polymerase can copy each strand
  from the point of the primer.
• Summary
• PCR requires the following-
• Template DNA
• Primers starting points for the construction
  of new strands
• Taq Polymerase a polymerase enzyme which works
  at high temperatures
• Supply of nucleotides
• PCR can amplify a single strand of DNA by a
  factor of millions

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This is a thermocycler which carries out the
process of PCR automatically by adjusting the
temperature and adding the ingredients when
required