Transcript Polymerase Chain Reaction

Polymerase Chain Reaction
Group 3:
Mitika Patel
Sheena Jain
Poonum Bharal
Aditi Dhakar
It is hard to exaggerate the impact
of the polymerase chain reaction.
PCR, the quick, easy method for
generating unlimited copies of any
fragment of DNA, is one of those
scientific developments that actually
deserves timeworn superlatives like
"revolutionary" and "breakthrough."
- Tabitha M. Powledge
Purpose of PCR
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Amplify specific nucleic acids in vitro
(“Xeroxing” DNA)
PCR will allow a short stretch of DNA (usually
fewer than 3000 base pairs) to be amplified to
about a million fold
This amplified sample then allows for size
determination and nucleotide sequencing
Introduced in 1985 by Kary Mullis
Millions of copies of a segment of DNA can be
made within a few hours.
Three Steps
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Separation: Double Stranded DNA is denatured by
heat into single strands.
Short Primers for DNA replication are added to
the mixture.
DNA polymerase catalyzes the production of
complementary new strands.
Copying The process is repeated for each new
strand created
All three steps are carried out in the same vial but
at different temperatures
Step 1: Separation
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Combine Target Sequence, DNA primers
template, dNTPs, TAQ Polymerase
 Target Sequence: Usually fewer than 3000 bp
– Identified by a specific pair of DNA primers- usually
oligonucleotides that are about 20 nucleotides
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Heat to 95 degrees Celsius to separate strands (for
0.5-2 minutes)
– Longer times increase denaturation but decrease
enzyme and template
Magnesium as a Cofactor
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Stabilizes the reaction between:
– oligonucleotides and template DNA
– DNA Polymerase and template DNA
 Heat Denatures DNA by uncoiling the
Double Helix strands.
Step 2: Priming
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Decrease temperature by 15-25 degrees
 Primers anneal to the end of the strand
 0.5-2 minutes
 Shorter time increases specificity but
decreases yield
 Requires knowledge of the base sequences
of the 3’ - end
Selecting a Primer
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Primer length
 Melting Temperature (Tm)
 Specificity
 Complementary Primer Sequences
 G/C content and Polypyrimidine (T, C) or polypurine
(A, G) stretches
 3’-end Sequence
 Single-stranded DNA
Step 3: Polymerization
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Since the Taq polymerase works best at
around 75 degrees C (the temperature of the
hot springs where the bacterium was
discovered), the temperature of the vial is
raised to 72-75 Degrees Celsius
The DNA polymerase recognizes the primer
and makes a complementary copy of the
template which is now single stranded.
Approximately 150 nucleotides/sec
Potential Problems with Taq
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Lack of proof-reading of newly synthesized DNA.
 Potentially can include diNucleotriphosphates
(dNTPs) that are not complementary to the
original strand.
 Errors in coding result
 Recently discovered thermostable DNA
polymerases, Tli and Pfu, are less efficient, yet
highly accurate.
Amplification
PCR Applications
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Detection of infectious diseases
 Detection of variations and mutations in
genes
 Detection of diseases from the past
 PCR and the law
Detection of infectious
diseases
- AIDS Virus
- Otitis Media-middle ear infection
- Lyme Disease-joint inflammation from tick
bites
- Detect 3 sexually transmitted diseases in one
swab-herpes, papillomarvirus, chlamydia
-Test to see if mother and baby have compatible
blood group-saves lives of babies
Detection of Variations and
Mutations in Genes
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Detects people with inherited disorders
 Lets us know who carries deleterious
variations (mutations)
 Direct way of distinguishing among the
confusion of different mutations in a single
gene. Ex: Duchenne muscular dystrophy
 Track presence or absence of DNA
abnormalities characteristic to cancer
Detection of diseases from the
past
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Presidential candidate Humphreys-had
cancer
 John Dalton-was colored blind and realized
that this was the case because he lacked a
gene for one of the three photopigments,
which caused him to be color blind
PCR and the Law
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DNA fingerprinting
– Can multiply small amounts of DNA found in blood
samples, hair, semen, and other body fluids
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Proving innocence of those already convicted
– Kirk Bloodsworth-wrongly accused of raping and
murdering a nine year old. Using PCR, he was proved
innocent and released from prison in 1993
Future of PCR:
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Copying larger pieces of DNA
 Miniaturization of hardware (chip-sized
devices)
 Computer automated test and analysis
 Taking PCR on the road and getting on the
spot DNA analysis
 Diagnose infection or genetic disorder right
in the doctors office
References
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“Polymerase Chain Reaction-Xeroxing DNA”
http://www.accessexcellence.org/AB/IE/PCR_Xeroxing_DNA.html
“The Polymerase Chain Reaction”
http://avery.rutgers.edu/WSSP/StudentScholars/project/archives/onions/pcr.html
“Polymerase Chain reaction” http://www.tulane.edu/~wiser/methods/handouts/pcr.PDF
Diagrams from : http://allserv.rug.ac.be/~avierstr/principles/pcrani.html
Purves, Sadava, Orians, Heller. “Life.” 6th ed. Sinauer Associates, 2001.
“Mechanism of PCR.” http://usitweb.shef.ac.uk/~mba97cmh/tutorial/pcr.htm
“The polymerase Chain Reaction”www.faseb.org/opar/bloodsupply/pcr.html