Polymerase chain reaction - Pcr

Ph¶n øng chuçi/d©y chuyÒn (cña/b»ng/nhê/do) Polymerase Polymerase Chain Reaction (PCR)Kary MullisGi¶i Nobel vÒ ho¸ häc, 1993 K.B. (1990) The unusual origin of the polymerase chain reaction. Scientific American. 262 (4) 56-65. devised by Kary Mullis c1983POLYMERASE CHAIN REACTION - PCRA 'licence' to do molecular biologyA key central technique that has revolutionised molecular and consequently cell biologySchematic illustration of PCR stepsMinh ho¹ b»ng s¬ ®å c¸c b­íc PCR From: Recombinant DNA by Watson, Gilman, Witkowski & ZollerTarget AmplificationNo. of	No. Amplicon Cycles	Copies of Target1	22	43	84	165	326	6420	1,048,57630	1,073,741,8241 cycle = 2 Amplicon2 cycle = 4 Amplicon3 cycle = 8 Amplicon4 cycle = 16 Amplicon5 cycle = 32 Amplicon6 cycle = 64 Amplicon7 cycle = 128 AmpliconPOLYMERASE CHAIN REACTIONThis lecture:Principles of PCR and applicationsPCR - the basicsHow to optimise PCR and troubleshoot problemsA 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 manipulationHuman diploid cell contains 6 X 10-9 base pairs 'average' gene size ~ 10,000bp = 1/300,000 600bp fragment = 1/1,000,000Amplification in a normal PCR will be perhaps a million foldWHAT IS PCR?Cloning of genes or gene fragmentssame species or homologous genes from different species (DOP-PCR)Genetic diagnosis - Mutation detectionbasis for many techniques to detect gene mutations (sequencing) - 1/6 X 10-9 bpPaternity testingMutagenesis to investigate protein functionQuantitate differences in gene expressionReverse transcription (RT)-PCRIdentify changes in expression of unknown genesDifferential display (DD)-PCR Forensic analysis at scene of crimeIndustrial quality controlAPPLICATIONS OF PCR Requires the binding of short sequences of DNA (oligonucleotides/amplimers/primers) to complementary sequences flanking the desired target regionthe 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°CEXTENSION 72°C25-35 CYCLESDENATURATION 93°C - 95°C DENATURATION 93°C - 95°C Denaturation; 	93°C - 95°C 30 secs – 1minAnnealing; 	37°C - 65°C30 secs – 1min depends on the melting temperature of duplexExtension/Polymerisation; 	72°C1min (+ 30secs per 500bp DNA)EACH PCR CYCLE HAS THREE STEPSCOMPONENTVOLUMEFinal Concentration10 X PCR Buffer5l1X10 X dNTPs (2mM)5l200MForward primer (10pmols/l)5l1M (50pmols/50l)Reverse primer (10pmols/l)5l1M (50pmols/50l)Genomic DNA template2l1gThermostable polymerase (2U/l)0.5l1 unitH2O (to 50l Final volume)27.5l TYPICAL REACTION MIXTURE25 or 50ls in a micro Eppendorf (0.5ml) tube CYCLING PARAMETERSDenaturation; 	93°C - 95°C 	30 secs – 1minAnnealing; 	37°C - 65°C	30 secs – 1min depends on the duplexExtension; 	72°C	1min 	(+ 30secs per 500bp DNA)25-35 cyclesFinal extension	2-10minsPCRAgarose gel electrophoresisThe final productUV visualisation3-4 hoursALWAYS REMEMBER!PCR is a highly sensitive technique – contamination with unwanted DNA can be a problemAlways run NEGATIVE controls Include a positive control if appropriateUse dedicated filtered tips and positive displacement pipettesDedicated areas?Can use UV cabinetsThe specific method should be ROBUST Re-optimise for each set of primersOPTIMISE PCR CONDITIONS AT THE OUTSETX 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	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 COMPONENTSTissue, cells, blood, hair root, saliva, semenObtain the best starting material you can.Some can contain inhibitors of PCR, so they must be removed e.g. Haem in bloodGood quality genomic DNA if possibleBlood – consider commercially available reagents Qiagen– expense?Empirically determine the amount to addTHE RAW MATERIAL 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 COMPONENTSNumber of options available	Taq polymerase	Pfu polymerase	Tth polymeraseHow big is the product?100bp 40-50kbWhat is end purpose of PCR?Sequencing - mutation detectionNeed high fidelity polymeraseintegral 3’ 5' proofreading exonuclease activity Cloning (TA cloning?)	CHOOSE YOUR POLYMERASE WITH CARET T TAAPCR productpGEM-T pCR 2.1-TOPO Taq - 	yesPfu -	noTA CLONING OF PCR PRODUCTS REQUIRES As 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 COMPONENTSLength ~ 18-30nt (21nt)Base composition; 50 - 60% GC rich	pairs should have equivalent TmsTm = [(number of A+T residues) x 2 °C] + 	[(number of G+C residues) x 4 °C]Initial use Tm–5°CAvoid internal hairpin structures	no secondary structureAvoid a T at the 3’ endAvoid overlapping 3’ ends – will form primer dimersCan modify 5’ ends to add restriction sites etcPRIMER DESIGN IS VITALPRIMER DESIGNUse specific programsOLIGOMedprobePRIMERDESIGNERSci Ed softwareAlso available on the internet 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 COMPONENTSTITRATE YOUR Mg2+ CONCENTRATION! 1 1.5 2 2.5 3 3.5 4 mMNormally, 1.5mM MgCl2 is optimal Best supplied as separate tubeAlways vortex thawed MgCl2Mg2+ concentration will be affected by the amount of DNA, primers and nucleotidesUSE MASTERMIXES WHERE POSSIBLETaken from -“ALL BLOCKS AND TUBES ARE EQUAL BUT SOME ARE MORE EQUAL THAN OTHERS!”G. Orwell (not!)Taken from - PERFECT RESULTIf nottroubleshootQiagen PCR methodsADDITIVES?Depends on the PCRCan be used where products are diffuse or absentDMSOFormamideGlycerolQIAGEN – QStratagene - Perfect Match®Stoffel fragmentVent™Deep Vent™PfuTthULTma™95 °C half-life40 min80 min400 min1380 min>120 min20 min>50 min5'3' exo++3'5' exo++++Extension rate (nt/sec)75>50>80?60>33?RT activityWeakWeak???Yes?Resulting ends3' A3' A>95% blunt>95% bluntblunt3' AbluntStrand displacement++M.W. (kDa)9461??929470Many useful sites:PCR Jump Station ON THE NET