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Lecture 11 Chapter 05-DNA-Amplification

PCR is a technique that amplifies a specific region of DNA using oligonucleotide primers and DNA polymerase. DNA is denatured and primers anneal to the target region. DNA polymerase then synthesizes new DNA strands that are complementary to the template strands. This results in exponential amplification of the target DNA region.

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12 views

Lecture 11 Chapter 05-DNA-Amplification

PCR is a technique that amplifies a specific region of DNA using oligonucleotide primers and DNA polymerase. DNA is denatured and primers anneal to the target region. DNA polymerase then synthesizes new DNA strands that are complementary to the template strands. This results in exponential amplification of the target DNA region.

Uploaded by

aridamansingh700
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© © All Rights Reserved
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Download as PDF, TXT or read online on Scribd
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PCR - Polymerase Chain Reaction

• PCR is an in vitro technique for the amplification of a region of DNA


which lies between two regions of known sequence.
• PCR amplification is achieved by using oligonucleotide primers.
– These are typically short, single stranded oligonucleotides which are
complementary to the outer regions of known sequence.

• The oligonucleotides serve as primers for DNA polymerase and the


denatured strands of the large DNA fragment serves as the template.
– This results in the synthesis of new DNA strands which are
complementary to the parent template strands.
– These new strands have defined 5' ends (the 5' ends of the
oligonucleotide primers), whereas the 3' ends are potentially
ambiguous in length.
Primer selection
• Primer is an oligonucleotide sequence – will target
a specific sequence of opposite base pairing (A-T,
G-C only) of single-stranded nucleic acids

• For example, there is a


– ¼ chance (4-1) of finding an A, G, C or T in any given DNA
sequence; there is a
– 1/16 chance (4-2) of finding any dinucleotide sequence (eg.
AG); a
– 1/256 chance of finding a given 4-base sequence.
• Thus, a sixteen base sequence will statistically be
present only once in every 416 bases (=4 294 967
296, or 4 billion): this is about the size of the human or
maize genome, and 1000x greater than the genome
size of E. coli.
Primer Specificity
• Universal – amplifies ALL bacterial DNA
for instance
• Group Specific – amplify all denitrifiers for
instance
• Specific – amplify just a given sequence
Forward and reverse primers
• If you know the sequence targeted for
amplification, you know the size which the
primers should be anealing across
• If you don’t know the sequence… What
do you get?
DNA Polymerase
• DNA Polymerase is the enzyme responsible for
copying the sequence starting at the primer from
the single DNA strand
• Commonly use Taq, an enzyme from the
hyperthermophilic organisms Thermus aquaticus,
isolated first at a thermal spring in Yellowstone
National Park
• This enzyme is heat-tolerant  useful both because
it is thermally tolerant (survives the melting T of
DNA denaturation) which also means the process is
more specific, higher temps result in less mismatch
– more specific replication

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