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Introduction to Polymerase Chain Reaction (PCR)
- 2. PCR : A brief history 1869 – DNA first isolated 1985 – In vitro amplification of DNA achieved using E.coli DNA polymerase 1988 – In vitro DNA amplification using Taq DNA polymerase 1989 – Taq DNA polymerase cloned and expressed in E.coli
- 3. • The Polymerase Chain Reaction (PCR) was not a discovery, but rather an invention • A special DNA polymerase (Taq) is used to make many copies of a short length of DNA (100-10,000 bp) defined by primers • Kary Mullis, the inventor of PCR, was awarded the 1993 Nobel Prize in Chemistry
- 5. PCR work was first published (1985)using Klenow polymerase – unstable with heat New enzyme had to be added manually at each step Maximum length 400bp Great idea – not very practical First reports using DNA polymerase from Thermus aquaticus (1988) Taq-polymerase (Saiki et al, 1988) from Yellow stone National Park hot springs Developed automatic “thermocycler” programmable heat block Development….
- 7. Polymerase Chain Reaction (PCR) • PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested. • The purpose of a PCR is to make a huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.
- 9. What all PCR Can Do ? •Starting with one original copy an almost infinite number of copies can be made using PCR •“Amplified” fragments of DNA can be sequenced, cloned, probed or sized using electrophoresis •Defective genes can be amplified to diagnose any number of illnesses •Genes from pathogens can be amplified to identify them (i.e., HIV, Vibrio sp., Salmonella sp. etc.) •Amplified fragments can act as genetic fingerprints
- 10. PROCEDURE …..
- 11. PCR Reagents 1X Buffer 10mM Tris-HCl, 50mM KCl MgCl2 1mM - 4mM (1.5mM) dNTPs 200μM Primers 100nM-1μM, 200nm (or less) for real time analysis DNA polymerase Taq DNA polymerase is thermostable 1-4 Units (1 unit) DNA 10pg-1μg (20ng)
- 12. Templates for PCR Dried blood Semen stains Vaginal swabs Single hair Fingernail scrapings Insect in amber Egyptian mummies Buccal swab Toothbrushes
- 13. DNA Replication Initiation - Forming the Replication Eye 3’ 5’ 3’ 5’ 5’ 5’ 3’ 3’ Origin of Replication 5’ 3’ 3’ 5’ 5’ 3’ 5’ 5’ 5’ 3’ 3’ 3’
- 14. Leading Strand Laging Strand 3’ 5’ 3’ 5’ Extension - The Replication Fork (Replication) 5’ 5’ 5’ 3’ 3’ 5’ 3’ 3’ 5’ Single strand binding proteins DNA Polymerase Okazaki fragment RNA Primers Primase 5’ 3’ 5’ Helicase 5’
- 15. How are the functions of replication achieved during PCR ??? . N/A as fragments are short Joining nicks . Taq Polymerase Polymerizing DNA . Primers added to the reaction mix Providing primer PCR Function . Heat Melting DNA ENZYMES • Helicase •SSB proteins •Topoisomerase •DNA pol •Primase •Ligase
- 16. PCR cycle & temperature profile Image source: Gene cloning and DNA analysis byTA brown, 6 edition, page no. 149, 152
- 17. PCR Melting 94 oC Temperature 100 0 50 T i m e 5’ 3’ 3’ 5’
- 18. PC R Melting 94 oC Temperature 100 0 50 T i m e 3’ 5’ 5’ 3’ Heat
- 19. Melting 94 oC Annealing Primers 50 oC Extension 72 oC Temperature 100 0 50 T i m e 3’ 5’ 5’ 3’ 5’ 5’ Melting 94 oC PC R
- 20. Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oC Temperature 100 0 50 T i m e 30x 3’ 5’ 5’ 3’ Heat Heat 5’ 5’ 5’ P C R
- 21. Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oC Temperature 100 0 50 T i m e 30x 3’ 5’ 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ P C R
- 22. Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oC Temperature 100 0 50 T i m e 30x 3’ 5’ 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ Heat Heat P C R
- 23. Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oC Temperature 100 0 50 T i m e 30x 3’ 5’ 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’
- 24. Fragments of defined length PCR Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oC Temperature 100 0 50 T i m e 30x 3’ 5’ 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’
- 25. More Cycles = More DNA Number of cycles 0 10 15 20 25 30 Size Marker
- 26. In summary • Primer length should not exceed 30 mer. • Tm, not more than 60 degree . • GC Content should be in the range of 40-60 % for optimum PCR efficiency. • Primers should end (3′) in a G or C, or CG or GC: this prevents “breathing” of ends and increases efficiency of priming.