Polymerase Chain Reaction Group 3: Mitika Patel Sheena Jain Poonum Bharal Aditi Dhakar.
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Transcript of Polymerase Chain Reaction Group 3: Mitika Patel Sheena Jain Poonum Bharal Aditi Dhakar.
PPolymerase olymerase CChain hain RReactioneaction
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 PCRPurpose 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 StepsThree 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: SeparationStep 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 CofactorMagnesium 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: PrimingStep 2: Priming
Decrease temperature by 15-25 degreesPrimers anneal to the end of the strand0.5-2 minutesShorter time increases specificity but
decreases yieldRequires knowledge of the base sequences
of the 3’ - end
Selecting a PrimerSelecting 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: PolymerizationStep 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 TaqPotential 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 ApplicationsPCR Applications
Detection of infectious diseasesDetection of variations and mutations in
genesDetection of diseases from the pastPCR and the law
Detection of infectious Detection of infectious diseasesdiseases
- 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 Detection of Variations and Mutations in GenesMutations in Genes
Detects people with inherited disordersLets 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 Detection of diseases from the pastpast
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 LawPCR 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:Future of PCR:
Copying larger pieces of DNAMiniaturization of hardware (chip-sized
devices)Computer automated test and analysisTaking PCR on the road and getting on the
spot DNA analysisDiagnose infection or genetic disorder right
in the doctors office
ReferencesReferences
“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