PCR理论及操作

PCR stands for “Polymerase Chain Reaction”, a process to replicate DNA.Polymerase chain reaction (PCR) is a fast and inexpensive technique used to amplify, or make many copies of,small segments of DNA.PCR is a technique used to make numerous copies of [i.e. amplify] a specific segment of DNA. PCR makes it possible to quickly and accurately obtain large quantities of DNA needed in carrying out research in molecular biology, in clinical diagnosis, in criminal investigations requiring forensic analysis, and in virus infectious disease research, e.g. in the ongoing battle against AIDS.PCR is to amplify DNA segment whose base sequence located its two ends have been cleared.Double stranded DNA.⏹PCR consists of three steps:PCR cycleDenaturationAnnealingExtension⏹The PCR stages are repeated for many cycles.The template DNA is separated into single strands by heating to 94℃for 1 minut .•When the temperature is reduced to around 54℃, the primers will bind to their complementary sequences. The primers binding is annealing.The temperature is increased to 72℃for the polymeraseto extend the primers into new complementary strands,which uses up dNTPs and required Mg++.30-40 cycles of 3 stepsWhen one PCR cycle is over, the target DNA will increase two times. Repeated denaturation, annealing and extension cycles multiply the target DNA exponentially.⏹First to know PCR principle, then prepare reaction mixture,finally program PCR procedure.⏹PCR amplification of a template requires two oligonucleotide primers, the fourdNTPs, magnesium ions, and a thermostable DNA polymerase to perform DNA synthesis.⏹PCR is a process based on the ability of a DNA polymerase enzyme that cansynthesize a complementary strand to a targeted segment of DNA in a test tubemixture of the four DNA bases.⏹(1) The mixture is first heated to denature (separate) the sides of the double-strandedDNA and then cooled to allow: (2) the primers to find and bind to their complementary sequences on the separated strands ;(3) the polymerase to extend the primers into new complementary strands.⏹Repeated heating and cooling cycles multiply the target DNA exponentially, since eachnew double strand separates to become two templates for further synthesis.In about 1 hour, 20 PCR cycles can amplify the target by a millionfold. In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created.Primary components of PCR mixture:⏹模板（template）⏹引物（primers）⏹DNA聚合酶（DNA polymerase）⏹PCR缓冲液（10×PCR buffer）⏹脱氧核苷三磷酸(dNTPs)⏹Mg++ (magnesium ions)⏹Sterile deionized water(灭菌去离子水）Template⏹The DNA or RNA tested in cells⏹When your vials (瓶）of cells arrive at the lab for testing, they are first mixed with adetergent which causes the cells to burst open and release their DNA along with other cell contents.⏹The mixture is then washed with a phosphate containing buffer (mild salt) solution todilute稀释cellular debris残渣. With minimal preparation, the sample样品is ready and the DNA on the targeted area of a chromosome can be amplified.What are primers ?⏹PCR mixture must also contain two DNA fragments, each about 20 bases long, calledprimers, that have sequences complementary to areas adjacent to each sides of the target sequence.⏹(To do PCR, you need to know the DNA sequence around the region you want toamplify.)⏹These primers can be constructed in the lab, or purchased from commercial suppliers. Ifchosen well, the 20-25 base pair sequence will be unique in the entire template so will match only the place specifically chosen thus limiting and defining the area to be copied. Why important are primers ?⏹Though DNA polymerase can elongate a polynucleotide strand by adding newnucleotides, it cannot start a strand from scratch头because it can only bond new nucleotides to a free sugar (3') end(-OH, hydroxy) of a nucleotide chain.⏹DNA polymerase requires the assistance of a primer, a previously existing short strand ofDNA (or RNA) that is complementary to the first part of the DNA segment being copied.⏹This small strand of nucleotides anneals (binds) by complementary base pairing to thebeginning of the area being copied.⏹With the primer in place, DNA polymerase is then able to continue adding the rest of thepairs of the segment until a new double strand of DNA is completed.⏹Primers are formed from free nucleotides in the cell by enzymes called RNA primases.⏹Through some quirk巧合of evolution, DNA polymerase is only able to add nucleotidesto the 3' (sugar) end of a growing chain of DNA. Therefore, it is not able just to start right up building a complementary strand for a length of DNA that has been separated from its partner.⏹To solve this problem in the cell, an enzyme called a primase produces a short stringof nucleotides (usually 15-30) that are complementary to the first part of the segment of DNA that is being copied.⏹This string of nucleotides, called a primer , attaches to the beginning of thetemplate strand by base pairing.⏹DNA polymerase is then able to add the next complementary nucleotide to the free 3'end of the primer. From there it continues adding more complementary nucleotides to the template DNA until a new double strand of DNA is completed.⏹Dual Primers⏹The sides of a DNA molecule are antiparallel, that is, they run in oppositedirections.⏹------so that the two sides of a DNA chain are replicated from differentdirections. Since DNA polymerase operates from the 5' (phosphate) to the 3' (sugar) end, this means that two different DNA primers are needed-----one for each 5' end of side of the molecule.⏹It is this requirement for dual primers that makes it possible to precisely define the area ofa DNA molecule to be amplified by the PCR.⏹By using custom made primers with complementary sequences at, or adjacent to, thebeginning of the marker targeted for amplification, it is possible to specify where the new chain should begin.Where to stop⏹Of course, it is also necessary to tell the DNA polymerase where to stop as well as whereto begin.⏹ A second primer with sequences that are complementary to the end of the targetedsegment is added.Preparation of Reaction MixtureStandard PCR⏹With all the improvements in PCR, what is currently considered standard PCR?⏹PCR amplification of a template requires two oligonucleotide primers, the four dNTPs,magnesium ions, and a thermostable DNA polymerase to perform DNA synthesis.⏹The target region to be amplified should be between 150 and 400 nucleotides.⏹The quantities of oligonucleotide primers, dNTPs, and magnesium, and the final volume,may vary for each specific application.Preparation of Reaction Mixture⏹To perform several parallel reactions, we recommend the preparation of a mastermix containing water, buffer, dNTPs, primers and Taq DNA Polymerase in a single tube, which can then be aliquoted等分into individual tubes. MgCl2 and template DNA solutions are then added. This method of setting reactions minimizes the possibility of pipetting移液errors and saves time by reducing the number of reagent transfers.Reaction Mixture Set Up⏹Gently vortex涡旋and briefly centrifuge离心all solutions after thawing融化.⏹Add, in a thin-walled PCR tube, on ice:⏹for 50µlof reaction mixture取无菌（或0.2 ml）PCR管一支，加：⏹10⨯PCR缓冲液5μl⏹氯化镁（25mmol/L）4μl⏹4种ｄNTP混合液(2.5mmol/L) 2μl⏹3'端引物(10μmol/L) 1μl⏹5'端引物(10μmol/L) 1μl⏹Taq DNA多聚酶(5u/μl) 0.25μl⏹蒸馏水31.75μl⏹模板DNA 5μl50µl of reaction mixture⏹10X PCR buffer 1X5µl⏹ 2.5mM dNTP mix 0.2mM of each 2µl⏹Primer I 0.1-1µM 1µl⏹Primer II 0.1-1µM 1µl⏹Taq DNA Polymerase 1.25u/50µl 5u/µl 0.25µl⏹25mM MgCl2 1-4mM* 4µl⏹Template DNA 10pg-1µg* 100ng 1µl⏹Sterile deionized water –variable 35.75µlTable for selection of 25mM MgCl2 solution volume:⏹Final concentration of MgCl2 (? mM) in 50µl reaction mix, Volume of 25mMMgCl2 :⏹ 1.0 1.25 1.5 1.75 2.0 2.5 3.0 4.0 (µl)⏹Gently vortex the sample and briefly centrifuge to collect all drops from walls of tube.⏹Overlay the sample with half volume of mineral oil or add an appropriate amount of wax蜡. This step may be omitted if the thermal cycler is equipped with a heated lid.⏹Place samples in a thermocycler and start PCR.Let me explain for 50µl reaction mixThe components of a standard PCR protocol using Taq DNA polymerase are as follows:(1) 10×Enzyme-specific reactionbuffer: 5(microlitreμl)10—50 mM Tris-HCl, pH 7.5—9.06—50 mM KCl or (NH4)2S041.5—5.0 mM MgCl2 or MgSO4(2) 0.2mM of each dATP, dGTP, dCTP, anddY~P 2μl(3) 0.1—1.0uM of each oligonucleotideprimer 0.5-1μl(4) 2.0—2.5 units of a thermostableDNA polymerase 0.5μl(5) Nucleic acid template, 1O2~1O5copies 1-2μl(6) Distilled蒸馏water to 50 μLControls⏹Even with the best reagents, primers, and supplies, proper controls must be performed totest for PCR performance and presence of DNA contamination.⏹As a rule, there should be a panel of negative and positive control samples to assay theefficiency and cleanliness of the sample preparation and pre-PCR process.1:阴性对照; 2:单引物R; 3:单引物F; 4:双引物R和F; 5:DL2000 MarkerThe negative controls⏹The negative controls that are run alongside each set of samples should be constructed toassay for sample-to-sample contamination.⏹The negative controls should include all reagents except the template DNA.Positive Controls⏹The amount of target DNA in positive controls should be minimized for two reasons. Totest for robust amplification, only 10—104 copies of the target sequence should be included in the reaction to serve as a valid control.⏹Second, by limiting the amount of DNA, there is less chance of this material sewing as asource of contamination. When setting up the reactions, always pipette the template in the tubes designated as positive controls last.Choice of ProceduresFor any of these reactions to work, there is a need to establish conditions for selective amplification of the target sequence.The determination of the temperatures to be used at each step in the PCR cycle.⏹The length of the primer extension step are dependent on the base composition of thetarget sequence, the length of amplicon扩增子, and the annealing temperatures of the primers.Procedures⏹PCR循环为：95℃预变性2 min，⏹95℃变性30 s、⏹55℃复性30 s、⏹72℃延伸1 min，⏹30个循环，最后72 C 延伸7 min⏹10℃保存20 hrWhere does PCR be performed in ?⏹PCR process is performed on a PCR cycler or PCR machine. The PCR cycler heatsand cools the PCR mixture.Reading material on PCRDNA Replication:How DNA Makes Copies of ItselfHomework1. List primary components of PCR mixture.2. Explain PCR principle andspecialized terms like Template and Primer.3. Write a standard PCR protocol using Taq DNA polymerase.。