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
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
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
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
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
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
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
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
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
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
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

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
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
DNA fingerprinting
Can multiply small amounts of DNA found in blood samples, hair, semen, and other body fluids
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:
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
“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