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Polymerase Chain Reaction (PCR)
Transcript of Polymerase Chain Reaction - WordPress.com · Polymerase Chain Reaction Used in medicine, genetics,...
Polymerase Chain Reaction
(PCR)
Polymerase chain reaction (PCR) - technique that allows scientists to make millions of copies of desired gene fragment in hours
Won him Nobel Prize in Chemistry in 1992
Polymerase Chain Reaction
Why was this technique so important?
Developed by Dr. Kary Mullis in 1987
Dr. Kary Mullis
Polymerase Chain Reaction
Used in medicine, genetics, biotechnology and forensicsMummy of Pharaoh Ramses II
HIV Research
The Human Genome Project
Crime Scene Investigation
PCR allows scientists to perform genetic testing on incredibly tiny amounts of tissue - even a single cellPCR can even be used on tissue that has been degraded by environment
Polymerase Chain ReactionPCR is similar to DNA replication:
Table 1: DNA Replication and PCR DNA Replication PCR
Double-stranded DNA Separates
TopoisomeraseDNA helicase
High Temperature (94˚C - 96˚C)
Primers Added to Single Strands RNA Primers DNA Primers
(forward and reverse primer)
Complimentary Strand Synthesized
DNA polymerase III copies entire DNA strand
Taq polymerase copies DNA fragments
Result Copy of entire DNA molecule After 30 cycles, over a billion identical DNA fragments
Polymerase Chain ReactionSTEP 1: DNA sample is heated to 94˚C - 96˚C
3’
5’
5’
3’
Heating breaks hydrogen bonds that hold double stranded DNA together -
complimentary single strands
Polymerase Chain ReactionSTEP 2: DNA primers added. Cooled to 50˚C - 65˚C
3’
5’
5’
3’
2 primers (forward and reverse) - chosen to be complimentary to
opposite ends of target region being copied
target region
Taq polymerase joins at primers and moves along strands adding complimentary base pairs as it goes
Polymerase Chain Reaction
3’
5’
5’
3’
STEP 3: Taq Polymerase added. Heated to 72˚C
target Region
target region
Result is two incomplete copies of DNA, each containing target region
Cycle 1 Complete
Taq polymerase comes from bacterium Thermus aquaticus, found in hot springs - enzyme does not denature at 72˚ C
STEP 4: Repeat steps 1 - 3
Cycle 2
Polymerase Chain Reaction
After cycle 2 result is four incomplete copies of DNA, each containing target region
target Region
target region
target Region
target region
Cycle 3
STEP 5: Repeat
Polymerase Chain Reaction
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Polymerase Chain Reaction
Cycle: DNA copies: Target copies:1,073,741,824 1,073,741,764
PCR can take a single copy of DNA and amplify it to
over a billion copies in just a few hours
More than enough material
to do many different types of genetic testing
