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recombinant_dna_technology

recombinant_dna_technology

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Published by rengachen

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Published by: rengachen on Oct 19, 2008
Copyright:Attribution Non-commercial

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02/01/2013

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Recombinant DNA
Technology
Needles in Haystacks

\u2022How to find one gene in large genome? A gene
might be 1/1,000,000 of the genome. Three
basic approaches:

\u2022
1. Polymerase chain reaction (PCR). Make
many copies of a specific region of the DNA.
\u2022

2. cell-based molecular cloning: create and
isolate a bacterial strain that replicates a copy of
your gene.

\u2022

3. hybridization: make DNA single stranded,
allow double strands to re-form using a labeled
(e.g. radioactive) version of your gene to make it
easy to detect.

Polymerase Chain Reaction
\u2022Based on DNA polymerase creating a second strand of DNA.

\u2013Needs template DNA and two primers that flank the region to be
amplified. Primers are short (generally 18-30 bases) DNA
oligonucleotides complementary to the ends of the region being
amplified.

\u2013DNA polymerase adds new bases to the 3' ends of the primers to create
the new second strand.
\u2013go from 1 DNA to 2, then 4, 8, etc: exponential growth of DNA from this
region
\u2013A key element in PCR is a special form of DNA polymerase from
Thermus aquaticus, a bacterium that lives in nearly boiling water in the

Yellowstone National Park hot springs. This enzyme, Taq polymerase,
can withstand the temperature cycle of PCR, which would kill DNA
polymerase from E. coli.

\u2022advantages:
\u2013rapid, sensitive, lots of useful variations, robust (works even with partly
degraded DNA)
\u2022disadvantages:
\u2013Only short regions (up to 2 kbp) can be amplified.
\u2013limited amount of product made

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