分子生物学分子生物学 (16).pdf

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1、Chapter 7Basic Methods of Gene Structure and Expression Analysis7.2 Polymerase Chain Reaction（PCR）Polymerase Chain Reaction（PCR）1.Principle of PCR technology1.Principle of PCR technology2.Heat2.Heat-resistant DNA polymeraseresistant DNA polymerase3.PCR primers and design principles3.PCR primers and

2、design principles4.Optimization of PCR conditions4.Optimization of PCR conditionsDNA biosynthesis(DNA half-retention replication)7.2.1 Principle of PCR technologyInvention of PCR technologyInvention of PCR technologyIn 1971,Khorana et al.Proposed In 1971,Khorana et al.Proposed the in vitro amplifica

3、tion of the in vitro amplification of nucleic acids:After DNA nucleic acids:After DNA denaturation,hybridize with denaturation,hybridize with appropriate primers,extend the appropriate primers,extend the primers with DNA polymerase,and primers with DNA polymerase,and repeat this process to clone the

4、 repeat this process to clone the tRNA gene.tRNA gene.In the spring of 1983,Kary Mullis proposed the idea of repeated DNA replication:heating denatures double-stranded DNA templates into single strands,cooling to combine single stranded DNA with primers(annealing),and adding DNA polymerase extension

5、 primers to synthesize new DNA.Kary MullisKary MullisThe chemical nature of the primer:The chemical nature of the primer:SingleSingle-stranded oligonucleotide DNA or RNA stranded oligonucleotide DNA or RNA fragments.fragments.In 1993,Kary Mullis won the Nobel Prize in Chemistry.In 1993,Kary Mullis w

6、on the Nobel Prize in Chemistry.In March 1985,Cetus applied for a PCR patent.In March 1985,Cetus applied for a PCR patent.In December 1985,PCR experiment technology was published In December 1985,PCR experiment technology was published in Science.in Science.PCR principlePCR principleDenaturationexte

7、nsionAnnealingDNA templateDNA templatePrimersPrimersdNTPdNTPDNA DNA PolymerasePolymeraseMgMg2+2+PCR is an in vitro amplification technology that mimics the process of natural DNA replication and uses DNA polymerase to catalyze the synthesis of specific DNA fragments between a pair of primers.Main th

8、ermal cycling processes:denaturation,annealing and extension.The basic principle of PCR reaction5 Primer 15 Primer 2Cycle 2Cycle 15 5 5 5 5 5 Template DNA5 5 5 5 5 5 5 5 DNA templateDNA templatePrimersPrimersdNTPdNTPDNA polymeraseDNA polymeraseMg2+Mg2+Principles of PCR technologyPrinciples of PCR te

9、chnologyCycle 35 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 PCR PrimersPCR Primers：Single-stranded DNA fragments provide 3-OH for DNA replication initiation.Cycle OneExtension products that are longer than the defined regions of the two primers.Only occurs in the amplification process using the original DNA as a

10、 template,amplified in multiples(2n).Primer extension productPrimer extension productCycle TwoPCR amplification final product sizePCR amplification final product sizeA A doubledouble-strandedstranded DNADNA fragmentfragment betweenbetween thethe 5 5 endsends ofof twotwoannealingannealing primersprim

11、ers.Cycle Threeplateau effect：After a certain number of cycles of PCR,After a certain number of cycles of PCR,DNA fragments no longer accumulate DNA fragments no longer accumulate exponentially,but enter a plateau period.exponentially,but enter a plateau period.Y=AY=A（1 1+E+E）n nY:Y:Amount of amplif

12、ication productAmount of amplification productA:A:Initial number of target DNA Initial number of target DNA moleculesmoleculesE:PCR amplification efficiencyE:PCR amplification efficiencyn:Nubbers of cn:Nubbers of cyclesyclesThe amount of PCR amplification productsThe amount of PCR amplification prod

13、uctsTaqDNA polymase5 3 polymerase activity；5 3 exonuclease activity；Lack of 3 5 exonuclease activity.It is isolated directly from Thermus aquatics It is isolated directly from Thermus aquatics YTYT-1 strain with high thermal stability.1 strain with high thermal stability.95 95 halfhalf-lifelife:40 m

14、in:40 min Optimum temperatureOptimum temperature:72 :72 80 80 Catalytic reaction rate:150 Catalytic reaction rate:150 300 nucleotides/300 nucleotides/second/enzyme moleculesecond/enzyme molecule7.2.7.2.2 2 HeatHeat-resistantresistant DNA polymeraseDNA polymeraseAnalysis of PCR products by Analysis o

15、f PCR products by agarose gel electrophoresisagarose gel electrophoresis500bp400bp200bp300bp1000bp600bp700bp800bp900bpMarkersPCR productPCR productPCR thermal cyclingPCR thermal cycling：High temperature High temperature denaturationdenaturation（93939898）Low temperature annealingLow temperature annea

16、ling（37376565）Medium temperature Medium temperature extensionextension（70707575）Realization of PCR automationRealization of PCR automation1.The sequence of the primer and template should be completely complementary2.Avoid the formation of stable dimers or hairpins between primers3.Primers cannot ini

17、tiate DNA polymerization reactions(mismatches)at non-target sites of the template7.2.3 PCR primers and design principles4.Primer length:1030 nt5.Base distribution:A,T,G,C random distribution6.G+C content：40 60%5CACTGAACGTAGGCCT33GCAGGTTCAGTGACTTGCATCCGGATGCAAC5Specific amplification5CACTGAACGTAGGCCT

18、33GGATCTACATGCGACTTAATCCGGAGATACC5Error raised1.Primer and template sequences should be closely complementary2.2.Primers cannot initiate DNA Primers cannot initiate DNA polymerization(mismatch)at nonpolymerization(mismatch)at non-target target sites in the templatesites in the templateBase excision3

19、.Optimization of PCR conditions(1)(1)Taq Taq enzyme concentrationenzyme concentration：1.01.02.5 U/100 l2.5 U/100 l(2)dNTP(2)dNTPconcentrationconcentration：2020200 mol/L 200 mol/L(3)Mg(3)Mg2+2+concentrationconcentration：0.50.52.5 mmol/L 2.5 mmol/L(4)(4)Primer concentrationPrimer concentration：0.10.10

20、.5 mol/L0.5 mol/LPCR application examples1.Diagnosis of genetic diseases:sickle cell anemia6 Glu Val(GAG GUG)PCR amplification of 6 DNA region2.Diagnosis of infectious diseases:HBV and HIV3.Diagnosis of tumor molecular markers:bcr-abl4.Individual identification:paternity testing and criminal investigation5.Biological archaeology:Isolation of gene fragments from the remains of bees,termites,and weevils in amber 25Ma-135 million years agoThank You!Thank You!Thank You!