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