Kĩ thật sử dụng PCR

Phản ứng chuỗi/dây chuyền (của/bằng/nhờ/do) Polymerase Polymerase Chain Reaction (PCR) Polymerase Chain Reaction (PCR) Kary Mullis Giải Nobel về hoá học, 1993 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. devised by Kary Mullis c1983 POLYMERASE CHAIN REACTION - PCR A 'licence' to do molecular biology A key central technique that has revolutionised molecular and consequently cell biology Schematic illustration of PCR steps Schematic illustration of PCR steps Minh ho¹ b»ng s¬ ®å c¸c b íc PCR Minh ho¹ b»ng s¬ ®å c¸c b íc PCR From: From: Recombinant DNA Recombinant DNA by Watson, by Watson, Gilman, Witkowski & Zoller Gilman, Witkowski & Zoller Target Amplification No. of No. Amplicon Cycles Copies of Target 1 2 2 4 3 8 4 16 5 32 6 64 20 1,048,576 30 1,073,741,824 1 cycle = 2 Amplicon 2 cycle = 4 Amplicon 3 cycle = 8 Amplicon 4 cycle = 16 Amplicon 5 cycle = 32 Amplicon 6 cycle = 64 Amplicon 7 cycle = 128 Amplicon POLYMERASE CHAIN REACTION This lecture: Principles of PCR and applications PCR - the basics How to optimise PCR and troubleshoot problems 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 6 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 WHAT IS PCR? • Cloning of genes or gene fragments same species or homologous genes from different species (DOP- PCR) • 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 APPLICATIONS OF PCR • 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. HOW DOES PCR WORK? 5' 3' 5' 3' 5' 3' 3' 5' ANNEALING 37°C - 65°C EXTENSION 72°C 25-35 CYCLES DENATURATION 93°C - 95°C DENATURATION 93°C - 95°C 1. Denaturation; 93°C - 95°C 30 secs – 1min 2. Annealing; 37°C - 65°C 30 secs – 1min depends on the melting temperature of duplex 3. Extension/Polymerisation; 72°C 1min (+ 30secs per 500bp DNA) EACH PCR CYCLE HAS THREE STEPS [...]... 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,... MASTERMIXES WHERE POSSIBLE Taken from -http://info.med.yale.edu/genetics/ward/tavi /PCR. html “ALL BLOCKS AND TUBES ARE EQUAL BUT SOME ARE MORE EQUAL THAN OTHERS!” G Orwell (not!) Taken from -http://info.med.yale.edu/genetics/ward/tavi /PCR. html THE PERFECT RESULT Qiagen PCR methods If not………………………troubleshoot ADDITIVES? Depends on the PCR Can be used where products are diffuse or absent DMSO Formamide Glycerol... ? >95% blu nt >95% blu nt + 3' A >80 3' A Weak >50 Weak 75 + + ? ? 61 + + 60 >33 ? ? Yes ? blunt 3' A blunt 92 94 70 PCR ON THE NET Many useful sites: PCR Jump Station http://www.highveld.com /pcr. html http://www.protocol-online.net/molbio/ http://info.med.yale.edu/genetics/ward/tavi /PCR. html Additives http://taxonomy.zoology.gla.ac.uk/~rcruicks/additives.html ... Final extension 2-10mins PCR Agarose gel electrophoresis 3-4 hours The final product UV visualisation ALWAYS REMEMBER! PCR is a highly sensitive technique – contamination with unwanted DNA can be a problem Always run NEGATIVE controls Include a positive control if appropriate Use dedicated filtered tips and positive displacement pipettes Dedicated areas? Can use UV cabinets OPTIMISE PCR CONDITIONS AT THE... blood, hair root, saliva, semen Obtain the best starting material you can Some can contain inhibitors of PCR, so they must be removed e.g Haem in blood Good quality genomic DNA if possible Blood – consider commercially available reagents Qiagen– expense? Empirically determine the amount to add OPTIMISING PCR – THE REACTION COMPONENTS • Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, saliva,... should be ROBUST Re-optimise for each set of primers X √ 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) OPTIMISING PCR – THE REACTION COMPONENTS • Starting nucleic acid - DNA/RNA...TYPICAL REACTION MIXTURE 25 or 50µls in a micro Eppendorf (0.5ml) tube COMPONENT VOLUME Final Concentration 10 X PCR Buffer 5µl 1X 10 X dNTPs (2mM) 5µl 200µM Forward primer (10pmols/µl) 5µl 1µM (50pmols/50µl) Reverse primer (10pmols/µl) 5µl 1µM (50pmols/50µl) Genomic DNA template 2µl 1µg Thermostable polymerase (2U/µl)... add restriction sites etc PRIMER DESIGN Use specific programs OLIGO Medprobe PRIMER DESIGNER Sci Ed software Also available on the internet http://www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.html 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 . Schematic illustration of PCR steps Schematic illustration of PCR steps Minh ho¹ b»ng s¬ ®å c¸c b íc PCR Minh ho¹ b»ng s¬ ®å c¸c b íc PCR From: From: Recombinant DNA Recombinant DNA by Watson,. 128 Amplicon POLYMERASE CHAIN REACTION This lecture: Principles of PCR and applications PCR - the basics How to optimise PCR and troubleshoot problems A simple rapid, sensitive and versatile. 1/1,000,000 Amplification in a normal PCR will be perhaps a million fold WHAT IS PCR? • Cloning of genes or gene fragments same species or homologous genes from different species (DOP- PCR) • Genetic diagnosis