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P olymerase C hain R eaction

P olymerase C hain R eaction. Group 3: Mitika Patel Sheena Jain Poonum Bharal Aditi Dhakar.

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P olymerase C hain R eaction

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  1. Polymerase Chain Reaction Group 3: Mitika Patel Sheena Jain Poonum Bharal Aditi Dhakar

  2. 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

  3. 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.

  4. 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

  5. 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

  6. Magnesium as a Cofactor • Stabilizes the reaction between: • oligonucleotides and template DNA • DNA Polymerase and template DNA

  7. Heat Denatures DNA by uncoiling the Double Helix strands.

  8. 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

  9. 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

  10. 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

  11. 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.

  12. Amplification

  13. PCR Applications • Detection of infectious diseases • Detection of variations and mutations in genes • Detection of diseases from the past • PCR and the law

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

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