PCR, yog cov tshuaj tiv thaiv polymerase, uas yog hais txog qhov sib ntxiv ntawm dNTP, Mg2+, elongation yam thiab kev ua kom muaj zog ntxiv rau cov kab ke hauv qab catalysis ntawm DNA polymerase, siv cov niam txiv DNA ua tus qauv thiab cov primers tshwj xeeb raws li qhov pib ntawm kev txuas ntxiv, Los ntawm cov kauj ruam ntawm denaturation, annealing, extension, thiab lwm yam, cov txheej txheem ntawm in vitro replicating ntxhais strand DNA ntxiv rau niam txiv strand template DNA tau sai thiab tshwj xeeb amplify tej hom phiaj DNA nyob rau hauv vitro.
1. Kub Pib PCR
Lub sijhawm pib ntawm kev nthuav dav hauv cov qauv PCR tsis yog muab lub tshuab PCR rau hauv lub tshuab PCR, thiab tom qab ntawd qhov kev pab cuam pib ua kom nrov.Thaum lub kaw lus configuration tiav lawm, lub amplification pib, uas tej zaum yuav ua rau tsis yog ib qho amplification, thiab kub-pib PCR yuav daws tau qhov teeb meem no.
Qhov kub pib PCR yog dab tsi?Tom qab cov tshuaj tiv thaiv tau npaj, cov enzyme hloov kho tau tso tawm ntawm qhov kub thiab txias (feem ntau siab dua 90 ° C) thaum lub sij hawm pib cua sov ntawm cov tshuaj tiv thaiv los yog "kub pib" theem, kom cov DNA polymerase tau qhib.Lub sijhawm ua kom muaj tseeb thiab qhov kub thiab txias nyob ntawm qhov xwm txheej ntawm DNA polymerase thiab qhov kub pib hloov pauv.Txoj kev no tsuas yog siv cov kev hloov pauv xws li cov tshuaj tiv thaiv kab mob, kev sib raug zoo ligands, lossis cov tshuaj hloov pauv tshuaj los tiv thaiv kev ua haujlwm ntawm DNA polymerase.Txij li thaum cov kev ua ntawm DNA polymerase yog inhibited nyob rau hauv chav tsev kub, kub pib tshuab muab kev yooj yim rau kev npaj ntau PCR tshuaj tiv thaiv kab mob nyob rau hauv chav tsev kub yam tsis muaj kev txi rau qhov tshwj xeeb ntawm PCR cov tshuaj tiv thaiv.
2. RT-PCR
RT-PCR (Reverse transcription PCR) yog ib qho kev sim rau kev thim rov qab los ntawm mRNA rau hauv cDNA thiab siv nws ua tus qauv rau kev nthuav dav.Cov txheej txheem sim yog txhawm rau rho tawm tag nrho RNA hauv cov ntaub so ntswg lossis cov hlwb ua ntej, siv Oligo (dT) ua tus primer, siv rov qab transcriptase los ua ke cDNA, thiab tom qab ntawd siv cDNA ua qauv rau PCR amplification kom tau txais lub hom phiaj gene los yog kuaj cov noob qhia.
3. Fluorescent quantitative PCR
Fluorescent quantitative PCR (Real-time Quantitative PCR,RT-qPCR) yog hais txog cov txheej txheem ntawm kev ntxiv cov fluorescent pawg rau PCR cov tshuaj tiv thaiv kab mob, siv cov khoom sib txuam ntawm cov teeb liab fluorescent los saib xyuas tag nrho cov txheej txheem PCR nyob rau lub sijhawm, thiab thaum kawg siv tus qauv nkhaus kom muaj nuj nqis txheeb xyuas cov qauv.Feem ntau siv qPCR txoj kev suav nrog SYBR Green I thiab TaqMan.
4. Nested PCR
Nested PCR hais txog kev siv ob txheej ntawm PCR primers rau ob lub voj voog ntawm PCR amplification, thiab cov khoom lag luam amplification ntawm qhov thib ob yog lub hom phiaj gene fragment.
Yog tias qhov tsis sib haum xeeb ntawm thawj khub ntawm primers (sab nrauv primers) ua rau cov khoom tsis tshwj xeeb yuav tsum tau amplified, qhov muaj peev xwm ntawm tib lub cheeb tsam uas tsis yog tshwj xeeb tau lees paub los ntawm ob khub primers thiab txuas ntxiv mus rau amplify yog me me, yog li cov amplification los ntawm ob khub ntawm primers, qhov tshwj xeeb ntawm PCR tau raug txhim kho.Ib qho txiaj ntsig ntawm kev ua ob zaug ntawm PCR yog tias nws pab txhawb cov khoom lag luam txaus los ntawm kev txwv tsis pub pib DNA.
5. Touchdown PCR
Touchdown PCR yog ib txoj hauv kev los txhim kho qhov tshwj xeeb ntawm PCR cov tshuaj tiv thaiv los ntawm kev kho cov kab mob PCR.
Hauv touchdown PCR, qhov kub thiab txias rau thawj ob peb lub voj voog yog teem ob peb degrees siab tshaj qhov siab tshaj plaws annealing kub (Tm) ntawm cov primers.Kev kub siab dua annealing tuaj yeem txo qhov tsis yog qhov tshwj xeeb amplification, tab sis tib lub sijhawm, qhov kub siab dua annealing yuav ua rau muaj kev sib cais ntawm primers thiab lub hom phiaj sib lawv liag, ua rau txo PCR tawm los.Yog li ntawd, nyob rau hauv thawj ob peb lub voj voog, lub annealing kub feem ntau yog teem kom txo los ntawm 1 ° C ib lub voj voog kom nce cov ntsiab lus ntawm cov hom phiaj hauv lub cev.Thaum lub annealing kub yog txo mus rau qhov zoo tshaj plaws kub, lub annealing kub yog khaws cia rau cov seem mus.
6. Direct PCR
Direct PCR hais txog kev nthuav dav ntawm lub hom phiaj DNA ncaj qha los ntawm cov qauv yam tsis tas yuav tsum muaj kev sib cais ntawm nucleic acid thiab purification.
Muaj ob hom kev ncaj qha PCR:
Txoj kev ncaj nraim: coj tus qauv me me thiab ntxiv ncaj qha rau PCR Master Mix rau PCR kev txheeb xyuas;
Txoj kev tawg: tom qab kuaj cov qauv, ntxiv rau lysate, lyse tso tawm cov genome, noj me me ntawm lysed supernatant thiab ntxiv rau PCR Master Mix, ua PCR txheeb xyuas.Txoj hauv kev no ua kom yooj yim rau kev sim ua haujlwm, txo cov sijhawm ua haujlwm ntawm tes, thiab zam DNA poob thaum lub sijhawm ua kom huv.
7. TSI PCR
Gene splicing los ntawm overlap extension PCR (SOE PCR) siv primers nrog complementary xaus los ua PCR cov khoom tsim overlapping chains, yog li ntawd nyob rau hauv lub tom ntej no amplification cov tshuaj tiv thaiv, los ntawm kev ncua ntawm cov overlapping chains, sib txawv qhov chaw ntawm ib tug txheej txheem nyob rau hauv uas amplified fragments overlapped. thiab spliced ua ke.Lub tshuab no tam sim no muaj ob lub ntsiab lus qhia: kev tsim kho cov noob fusion;gene site-directed mutation.
8. PIB
Inverse PCR (IPCR) siv cov txheej txheem rov qab los ua kom zoo dua DNA cov seem uas tsis yog ob lub primers, thiab nthuav cov kab lus tsis paub txog ntawm ob sab ntawm cov kab DNA paub.
IPCR yog thawj zaug tsim los txiav txim qhov sib lawv liag ntawm cov cheeb tsam uas nyob ib sab tsis paub, thiab feem ntau yog siv los kawm txog cov noob caj noob ces;oncogenic chromosomal rearrangements, xws li gene fusion, translocation thiab transposition;thiab kis kab mob sib xyaw ua ke, kuj tau siv tam sim no Rau qhov chaw qhia mutagenesis, luam cov plasmid nrog rau qhov xav tau hloov pauv.
9. dPCR
Digital PCR (dPCR) yog cov txheej txheem rau kev ua kom muaj nuj nqis ntawm nucleic acid molecules.
Tam sim no muaj peb txoj hauv kev rau kom muaj nuj nqis ntawm nucleic acid molecules.Photometry yog raws li absorbance ntawm nucleic acid molecules;real-time fluorescent quantitative PCR (Real Time PCR) yog raws li tus nqi Ct, thiab tus nqi Ct yog hais txog lub voj voog tus lej sib raug rau tus nqi fluorescence uas tuaj yeem kuaj pom;digital PCR yog qhov tseeb Quantitative technology raws li ib leeg-molecule PCR txoj kev suav nucleic acid kom muaj nuj nqis yog ib txoj hauv kev kom muaj nuj nqis.
Post lub sij hawm: Jun-13-2023