分子生物学PCR

Polymerasechainreaction(PCR)PCR•PCRDNADNADNADNA2n•PCRPCR•PCR—94°C30sec—4555°C30sec—72°C2kb/minPCR••PCR(pg=10-12)(µg=10-6)•PCR24PCR••DNA•dNTP••Mg2+PCR•DNADNA5’5’PCR•PCR—1530————3’5’PCR•16bp30bp2024bp—5’•3’—3’2“”“”•G+C%—G+C%•PCR•0.11M••30PCR•—PCR—G+C%TaTm5°CTa=Tm-5°C=4(G+C)+2(A+T)-5°C—•—DNA7075°C—72°CDNA35100/1kbpHDNA—dNTP•dNTP50mMTaqDNA•dNTP•PCR50200M1015M•dNTPDNA•TaqDNA—PCR2.5U/——1041•DNA—DNA—PCRDNA•—DNARNA—RNAcDNA•—DNAngDNA—DNADNA—•—PCRDNADNAPCR——DNADNA—9095°CPCR•—1050mMTris–HClpH8.3—72pH17.2•—TaqDNAMg2+—Mg2+—1.5mmol/LPCR•PCR2540———•PCR—DNA—DNA2nDNA—DNA—PCR•PCR1050mmol/LTris-HCl50mmol/LKCl•1.5mmol/L•20200µmol/LdNTP•TaqDNA7080°C•0.10.5µmol/LPCR•—1010µl—4dNTP200µmol/L—10100pmol—DNA0.12µg—TaqDNA2.5u—Mg2+1.5mmol/L—100µl•—94°C/3060s—4555°C/3060s—72°C—253035•PCR———•—EP—PCR————MgCl2—PCR——Mg2+———————2C—PCRMg2+—PCR[Mg2+].—0.5mMPCR[Mg2+]—PCR—TaqDNA3’→5’—TaqDNA104—400bp2033DNA—PCR•PCR—DNADNADNA—PCRPCR—PCRPCRPCRPCRPCR•PCR—TaqDNADNA3’A:NNNNNNN…NNNNNNNAANNNNNNN…NNNNNNN—T/A(T/Acloning)—T-PCRPCRReversetranscriptionPCR(RT-PCR)•RNAmRNAolig-dTcDNAcDNAPCR•———cDNA—RNAoligo(dT)cDNAPCRPCR(nestedPCR)PCRP1P1P2PCRP1P2P2PCR•PCR•PCR(inversePCR)PCR•T-DNADNAPCR•PCRPCR•—PCRTaq5’3’DNAPCR—PCRSYBRSYBRDNASYBRPCR“Real-Time”PCRFQ…...…...T1.TaqPolymerase“finds”Theprimer2.Laserexcitesfluorescenttagonprimer3.Fluorescenceisabsorbedbyquenchermolecule4.Detectorregisters“noreaction”.5.PrimerisextendedbyTaqPolymeraseFQ…...…...T….6.ExtensioncontinuesuntilthePolymeraseMeetsthefluorescentmolecule.FQ…...…...T…...….7.Thepolymerasebreaksdownthetaggedprimer.Thefluorescenttagandquenchermoleculebecomeseparated.Q…...T…...…...…...…QFF8.AsPCRcontinues,thetaggedprimersaredegradedand“free”fluorescentmoleculesaccumulate.9.Thedetectorrecordsfluorescenceasameasureofamplificationprogress.QuantificationbyReal-TimePCR35SControl