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PowerPoint Presentation http //www nobel se/chemistry/laureates/1993/mullis autobio html Mullis, K B (1990) The unusual origin of the polymerase chain reaction Scientific American 262 (4) 56 65 devise[.]

POLYMERASE CHAIN REACTION - PCR A 'licence' to molecular biology A key central technique that has revolutionised molecular and consequently cell biology devised by Kary Mullis c1983 http://www.nobel.se/chemistry/laureates/1993/mullis-autobio.html Mullis, K.B (1990) The unusual origin of the polymerase chain reaction Scientific American 262 (4) 56-65 POLYMERASE CHAIN REACTION This lecture: Principles of PCR and applications PCR - the basics How to optimise PCR and troubleshoot problems WHAT IS PCR? A simple rapid, sensitive and versatile in vitro method for selectively amplifying defined sequences/regions of DNA/RNA from an initial complex source of nucleic acid - generates sufficient for subsequent analysis and/or manipulation Human diploid cell contains X 10-9 base pairs 'average' gene size ~ 10,000bp = 1/300,000 600bp fragment = 1/1,000,000 Amplification in a normal PCR will be perhaps a million fold APPLICATIONS OF PCR •Cloning of genes or gene fragments same species or homologous genes from different species (DOPPCR) •Genetic diagnosis - Mutation detection basis for many techniques to detect gene mutations (sequencing) - 1/6 X 10-9 bp •Paternity testing •Mutagenesis to investigate protein function •Quantitate differences in gene expression Reverse transcription (RT)-PCR •Identify changes in expression of unknown genes Differential display (DD)-PCR •Forensic analysis at scene of crime •Industrial quality control HOW DOES PCR WORK? •Requires the binding of short sequences of DNA (oligonucleotides/amplimers/primers) to complementary sequences flanking the desired target region •the action of an enzyme (DNA polymerase) to synthesise new exact copies of the target DNA 3' 5' 5' 3' 3' 3' 5' 5' DENATURATION 93°C - 95°C ANNEALING 37°C - 65°C 25-35 CYCLES DENATURATION 93°C - 95°C EXTENSION 72°C EACH PCR CYCLE HAS THREE STEPS Denaturation; 30 secs – 1min 93°C - 95°C Annealing; 37°C - 65°C 30 secs – 1min depends on the melting temperature of duplex Extension/Polymerisation; 72°C 1min (+ 30secs per 500bp DNA) TYPICAL REACTION MIXTURE 25 or 50ls in a micro Eppendorf (0.5ml) tube COMPONENT VOLUME Final Concentration 10 X PCR Buffer 5l 1X 10 X dNTPs (2mM) 5l 200M Forward primer (10pmols/l) 5l 1M (50pmols/50l) Reverse primer (10pmols/l) 5l 1M (50pmols/50l) Genomic DNA template 2l 1g Thermostable polymerase (2U/l) 0.5l unit H2O (to 50l Final volume) 27.5l CYCLING PARAMETERS Denaturation; 93°C - 95°C 30 secs – 1min Annealing; 37°C - 65°C 30 secs – 1min depends on the duplex Extension; 72°C 1min (+ 30secs per 500bp DNA) 25-35 cycles Final extension 2-10mins PCR Agarose gel electrophoresis 3-4 hours The final product UV visualisation OPTIMISING PCR – THE REACTION COMPONENTS • Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, saliva, semen • Thermo-stable DNA polymerase e.g Taq polymerase • Oligonucleotides Design them well! • Buffer Tris-HCl (pH 7.6-8.0) Mg2+ dNTPs (dATP, dCTP, dGTP, dTTP) CHOOSE YOUR POLYMERASE WITH CARE Number of options available Taq polymerase Pfu polymerase Tth polymerase •How big is the product? 100bp 40-50kb •What is end purpose of PCR? Sequencing - mutation detection Need high fidelity polymerase integral 3’ 5' proofreading exonuclease activity Cloning (TA cloning?) TA CLONING OF PCR PRODUCTS REQUIRES As A A PCR product Taq - yes T T pGEM-T pCR 2.1-TOPO Pfu - no OPTIMISING PCR – THE REACTION COMPONENTS • Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, saliva, semen • Thermo-stable DNA polymerase e.g Taq polymerase • Oligonucleotides Design them well! • Buffer Tris-HCl (pH 7.6-8.0) Mg2+ dNTPs (dATP, dCTP, dGTP, dTTP)