Polymerase Chain Reaction PCR

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Polymerase Chain Reaction (PCR)
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Polymerase Chain Reaction (PCR)

• What is PCR?
• PCR is a method by which a large number of copies
  of a specific gene, or DNA fragment, can be made
  from a very small amount of DNA.
• What is PCR used for?
• Clinically, PCR may be used to diagnose genetic
  disorders, aid in forensic sciences (eg. DNA
  fingerprinting), and detect infectious agents
  (eg. AIDS virus).

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How Does PCR work?

• Before PCR can begin, reagents must be added to
  the sample so that copies of the DNA can be made.
  This reagents include primers (oligonucleotides
  that target a specific DNA sequence), nucleotides
  (building blocks of new DNA) and Taq polymerase
  (an enzyme which builds the new DNA strands).
• PCR is carried out in an instrument called a
  thermocycler. The thermocycler carries the
  reaction through repeated cycles of DNA
  denaturation, annealing and polymerization,
  taking advantage of the chemical properties of
  DNA and the reagents at specific temperatures.

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PCR
3-5 Primer
DNA
5-3 Primer
Taq Polymerase
Free Nucleotides
Step 1Denaturation
5?
3?
3?
5?
The sample (genomic DNA or cDNA, made from RNA)
is heated to 94C. This temperature causes the
double stranded DNA to denature, or separate into
two single strands exposing the nucleotides which
make the bonds between the two strands.
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Step 2 Annealing
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The sample is cooled to 54C. The primers bind
the single stranded DNA at their complementary
sequences.
Step 3 Polymerization
The sample is heated back up to 72C. This is
optimal for the Taq polymerase to add on
nucleotides creating new DNA. When the sample is
heated back to 94C, it inactivates the
polymerase and the newly synthesized double
stranded product denatures, and the cycle is
repeated as many times as necessary (typically
35-40).
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Result
logarithic increases
cycle 3
cycle 1
cycle 2
Desired gene segment
Note that the first cycle will make DNA longer
than the distance between the primers but
subsequent cycles favor the synthesis of
sequences only between the primers because the
polymerase falls off the shorter DNA strand
cycle 4
The results can be visualized by gel
electrophoresis (or other means).
Reference ladder To ensure that the product you
are measuring is the right size (correct number
of base pairs)
Amplified product If a band is present then
sample is positive for DNA in question
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Variations

• Reverse Transcriptase PCR
• Can measure the expression of certain RNA
  sequences by converting the RNA to DNA before
  doing PCR
• Real-Time PCR
• Quantifies the product