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Lecture

Biology Chapter 19.docx


Department
Biology
Course Code
BIOLOGY 1A03
Professor
Lovaye Kajiura

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Biology Chapter 19: Analyzing and Engineering Genes
The Polymerase Chain Reaction
The polymerase chain reaction (PCR) is an in vitro DNA synthesis reaction in which a
specific section of DNA is replicated over and over to amplify the number of copies of
that sequence
It’s a technique for generating many identical copies of a particular section of DNA
Although PCR is much faster and technologically easier than cloning genes into a DNA
library, it is only possible when a researcher has some information about DNA
sequences near the gene in question
Sequence information is required because to do a polymerase chain reaction, you have
to start with short lengths of a single-stranded DNA that match sequences on either side
of the gene of interest
These short segments act as primers for the synthesis reaction
Primer sequences must be complementary to bases on either side of the gene in
question
One primer is complementary to a sequence on one strand upstream of the target DNA,
the sequence of interest
The other primer is complementary to a sequence on the other strand, downstream of
the target DNA
If the target DNA molecule is made single stranded, then the primers will bond or anneal
to their complementary sequences
Once the primers are bound, DNA polymerase can extend each strand in the 5’ to 3’
direction
A PCR procedure begins with a reaction mix containing an abundant supply of dNTP of
the template DNA, copies of the two primers and an enzyme called Taq polymerase
o Taq polymerase is a DNA polymerase found in the bacterium Thermus aquaticus,
which was originally discovered in a hot spring
o Taq polymerase is the enzyme of choice in PCR because the technique depends
on heating the reaction mix and because Taq polymerase is heat stable
o Taq polymerase continues to function normally even when heated to 95oC
PCR gets under way when the reaction mix is heated to 94oC
o At this temperature, the double-stranded template DNA denatures
o This means that the two DNA strands separate, forming single-stranded
templates
o Then, the mixture is allowed to cool to 50-60oC
o In this temperature range, some of the denatured DNA strands re-form double
helices
But some of the primers bond or anneal to complementary portions of the single-
stranded template DNA
o This step is called primer annealing
The reaction mix is then heated to 72oC
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