sequencing the polymerase chain reaction (pcr) and its ：测序聚合酶链反应（pcr）和

Adapted from a presentation written forPrinciples of Gene Manipulation November 6, 2000Jennifer Cooper America Madrigal Lalea Vellanoweth,AMPLIFICATION:,The Polymerase Chain Reaction,11.06.00,AMPLIFICATION: PCR and Its Applications,Definition of PCRRequirements for PCRPCR ProcessA. DenaturationB. Annealing C. ExtensionD. Cycling (repeat A-C)PCR for HLA DQ-alpha,11.06.00 2,11.06.00,PCR What is it?,The Polymerase Chain Reaction (PCR) is an in vitro method to amplify a specific region of DNA. PCR is extremely sensitive, with the capabilityof amplifying minuscule quantities of DNA.,11.06.00 3,11.06.00,PCR REQUIREMENTS,In the reactionSample templatePrimersHigh temperature resistant polymerase; e.g., TaqDeoxynucleotide triphosphates dNTPs (dATP, dGTP, dCTP, dTTP)BufferMg+, KClThermocycler instrument programmed to change samples rapidly from one set temperature to another,11.06.00 4,11.06.00,PCR METHOD,There are three basic steps in PCR1. Denaturation (95oC)2. Annealing (55oC, but varies)3. Extension (72oC)Cycling repeats Steps 1-3 up to 35 times.,11.06.00 5,11.06.00,PCR METHOD DENATURATION STAGE,11.06.00 6,(reference: /24355/data/light/details/media/polymeraseanim.html),High temperature separates the two strands.,PCR METHOD ANNEALING STAGE,11.06.00 7,(reference: /24355/data/light/details/media/polymeraseanim.html),Primer length is usually 20 nucleotides.,11.06.00,PCR METHOD EXTENSION STAGE,11.06.00 8,(reference: /24355/data/light/details/media/polymeraseanim.html),Thermostable polymerase adds dNTPs one at a time at this stage.,11.06.00,PCR METHOD - CYCLING,11.06.00 9,(reference: /24355/data/light/details/media/polymeraseanim.html),The average number of cycles = 30 for efficiency reasons. 230 = 1.07 X 109 copies,11.06.00,PCR METHOD - CYCLING,DNACycle 1 2 3 4 5 6 Products 2 4 8 16 32 ,11.06.00 10,After the first two cycles, fragments with the correct length begin to be amplified. (dictated by the placement of the primers),11.06.00,PCR for HLA DQ-alpha,Biotinylated primers amplify a region of 250 bp.Sequence of primers is common to all individuals.Sequence between primers is polymorphic may differ between chromosomes and among individuals.PCR picks out the 250 bp HLA DQ- region from 3.3 X 109 bp of DNA present in 23 chromosomes.Note that in the kit we are using there are actually six pairs of biotinylated primers (12 primers total).Only 1 pair will amplify the HLA DQ- region The other five pairs of primers amplify genes that we are not interested in for this class. The analysis for these genes employs a different probe strip than the HLA DQ- probe strip that we will be using.After agarose gel electrophoresis six bands will be seen, only one of which is the band of interest for this class.,11.06.00 11,11.06.00,The human male karyotype: 22 homologous chromosomes + X and Y,11.06.00,11.06.00,Products of PCR reaction 106 double strand fragments 250 bp in length.All are biotinylated (biotinylated primers). 1/2 = DQ- allele on one copy of your Chromosome 6; 1/2 = DQ- allele on the other copy.,PCR for HLA DQ-alpha,11.06.00,PCR REFERENCES,1. Gene Cloning: an introduction. T.A. Brown2. The world wide web: /PCR/whatisPCR.html/AB/GG/polymerase.html/1998/0598issue/0598working.html/opar/bloodsupply/pcr.html/donald.slish/PCR.html/24355/data/light/details/media/polymeraseanim.html,11.06.00 15,11.06.00,After a long process, this PowerPoint presentation has now ended. We hope it is of good help in your studies.,11.06.00,PCR RequirementsThe Details,11.06.00,PCR REQUIREMENTS: sample - template,AmountNeeded very small; intact DNA from one cellto see on a gel, need 1011 final copies; need 104 starting copiesConcern competition with primers for annealing byToo much starting templateToo much product from excessive cyclingRemember, the association rate of two strands increases with the square root of the length of the DNA. Longer strands anneal more quickly than shorter. So . . .Templates and products are longer than primers. In high concentration, they reanneal before primers can anneal.,11.06.00 4,11.06.00,PCR REQUIREMENTS: sample - template,Even degraded DNA is OK if sample is large enoughFossilsRemainsOld samples from crime scenes,11.06.00,PCR REQUIREMENTS - primers,Two primers of known sequenceflank region you are interested inanneal to opposite strands of templateprime toward the region between themnon-complementary to each otherlack internal complementarityof sufficient length to anneal to unique site in the genome (20 nt)1/420 = 1 site of identical sequence/1 X 1012 bpchance that any one of the 4 bases will be at a given site = 1/4have similar annealing temperaturespresent in excess (0.1 1.0 uM each)favor annealing of primer over reannealing of strandssufficient for amplification through 25-30 cycles,11.06.00,PCR REQUIREMENTS - dNTPs,must be present in sufficient excess to complete extension through all cycles (200 M in each dNTP)must not be present in such high excess that Mg+ ions are complexed and unavailable as cofactors for polymerase activity,11.06.00,PCR REQUIREMENTS - “buffer” components,The buffer, e.g., Tris base adjusted to a specific pH with HClmaintains pH pH 8.3 is optimum for Taq.pH optimum keeps protein folded in a conformation at which it is enzymatically active.Different temperature-insensitive polymerases have different pH optima,11.06.00,PCR REQUIREMENTS - “buffer” components,Monovalent salt, e.g., KClto contribute to correct folding of enzyme and thereby to contribute to optimum activity of enzyme,11.06.00,PCR REQUIREMENTS - “buffer” components,Mg+ (MgCl2, MgSO4)Mg+ is a cofactor for DNA polymerasesfor Taq, required free Mg+ = 2mMcalculated Mg+ may differ from the actual free Mg+Positively charged Mg+ is complexed by ionic bonding with the negative charges on primers, template, and dNTPsMg+ must be uncomplexed (free) to act as a cofactor for the polymerase,11.06.00,PCR REQUIREMENTS - “buffer” components,Mg+ (contd)Determining the optimal concentration of Mg+ is the most important step in setting up PCR conditionstoo little - polymerase cant worktoo much - favors annealing of primers to mismatched locations on the template,11.06.00,PCR REQUIREMENTS - polymerase,1-2 units of enzyme/100 l reactionHigh temperature resistantable to remain active through up to 35 cycles with DNA denaturation at 95oCExample: Taq polymeraseisolated from Thermophilus aquaticus,11.06.00,PCR REQUIREMENTS - polymerase (contd),Many different polymerases availablesome have both polymerase and editing (exonuclease) activities,Pfu polymerase can edit. Taq polymerase can not.Taq polymerase is more likely to misincorporate a dNTP.,11.06.00,PCR REQUIREMENTS - polymerase (contd),Many different polymerases availableleave different types of endsbluntsingle A 3overhangeach isolated from a different organism which has evolved to survive at high temperaturesdeep water vents hot springs,11.06.00,PCR REQUIREMENTS - denaturation of dsDNA,Denaturation temperaturehigh enough to overcome attractive energy of H-bonds between bases of the complementary template and product strands95oC provides sufficient energy to separate even long strands,11.06.00,PCR REQUIREMENTS - annealing,The lower the temperature, the easier it is for 2 strands of DNA to pair with each otherSo the chosen annealing temperature must beHigh enough to prevent hybridization of primers to imperfectly complementary template sequences (i.e., non-specific annealing)Not so high that the primers cant anneal to template DNA at all,11.06.00,PCR REQUIREMENTS - annealing,What determines the optimum annealing templength of primer - the longer, the higher the optimum annealing temp will belonger the primer, the more H-bondsSo, the more H-bonds, more likely 2 strands are to anneal or stay annealed% GC - the more GC, the higher the optimum annealing temp will beGC base pairs have 3 H-bonds; AT base pairs have only 2So, the more GC, more likely 2 strands are to anneal or stay annealedthe the salt, the the optimum annealing temp will bepositive ions in salt are counterions to the negatively charged sugar-phosphate backbone of the ds DNApositive counterions prevent repulsive forces of negative charges from pushing the strands apart,11.06.00,PCR REQUIREMENTS - annealing,Annealing temperature for PCR is often set at 5oC below the TmTm = temperature at which 50% of the possible correct primer/template complexes are unformedEstimate Tm for primers 10-23 nt long in 1M saltTm (oC) = 4 (G+C) + 2 (A+T),11.06.00,Hot start,Way to minimize early non-specific annealing that causesprimer dimersamplification of incorrect product,Why might early annealing be expected to be non-specific?,11.06.00,Hot start,Enzyme is not mixed with reaction until sample has reached denaturation temperature manual addition of enzyme at 95Cpolymerase separated from other reagents by layer of solid waxwax melts at denaturation temperature, polymerase mixes with reagents, wax rises to top and prevents evaporationstart with antibody/polymerase complex antibody denatured and releases enzyme at 95C,11.06.00,PCR REQUIREMENTS - extension,Note: the longer the expected product, the longer the extension time requiredexact time depends on rate of progression of the specific polymeraseExtension temperatureoptimal temperature for enzymedetermined for each enzyme empiricallyusually around 72oC,11.06.00,PCR REQUIREMENTS,Thermocycler - instrument programmed to change samples rapidly from one set temperature to another,11.06.00,PCR REQUIREMENTS,Way to prevent evaporation of water from reaction at high temperaturesWhy?evaporation raises concentrations of reaction solutes inhibition of reactionHow?Thermocycler applies heat to the top of the reaction tube, thereby preventing condensationor, overlayer the reaction with mineral oil, preventing evaporation,11.06.00,PCR REQUIREMENTS - thermocycler,Way to heat and cool the sampleSolid heating/cooling block that holds samplesEfficient conduction of heat between heating/cooling block and samplepressure applied from top pushes walls of tube directly against block, eliminating air space, ormineral oil is used to fill in air space between heating block and sample,11.06.00,PCR REQUIREMENTS - controls,No template controlshould be no productif there is, contaminating DNA is presentKnown positive (if possible)should be a producttells you all reaction components are workingmay tell you what your product should look like,11.06.00,PCR REQUIREMENTS - controls (contd),Size markers show what size your product isand if you know what size to expect, whether you are getting the expected productResults usually analyzed by gel electrophoresis,11.06.00,PCR REQUIREMENTS -Minimizing Contamination,Contamination of pipettorsUse aerosol barrier pipet tipsContamination of supplies and reagentsUV irradiation base/acid treatment of reusable supplies,11.06.00,PCR REQUIREMENTS -Minimizing Contamination (contd),Contamination of work area with sample or productPerform steps at separated benches or roomssample prepreaction set upthermocyclingproduct analysisPrevent aerosols containing PCR productscentrifuge reagents and products before opening tubealso prevents contamination of reagents from glovesuncap tubes carefully,11.06.00,PCR REQUIREMENTS Minimizing Contamination (contd),Contamination of reaction mixUse aerosol barrier pipettor tipsUse distilled deionized waterAdd DNA to the reaction last,11.06.00,PCR REQUIREMENTS -Troubleshooting,No yieldExtra or incorrect productsPrimer dimersMisincorporation,11.06.00,PCR REQUIREMENTS -Troubleshooting,No yieldWere all reagents included?Insufficient denaturation?Higher tempCheck conditions for transfer of heat from block to tube Active nucleases or proteases present in rx?Insufficient free Mg+?Bad primers?DegradedWrong sequence,11