PCR (polymerase saw cov tshuaj tiv thaiv) yog ib qho ntawm in-vitro DNA amplification technologies, nrog rau keeb kwm ntawm ntau tshaj 30 xyoo.

PCR thev naus laus zis tau pioneer los ntawm Kary Mullis ntawm Cetus, Tebchaws Asmeskas hauv xyoo 1983. Mullis tau thov rau PCR patent hauv xyoo 1985 thiab luam tawm thawj daim ntawv qhia txog PCR ntawm Science hauv tib lub xyoo.Mullis tau txais txiaj ntsig Nobel nqi zog hauv chemistry hauv 1993 rau nws txoj haujlwm.

Basic Principles ntawm PCR

PCR tuaj yeem nthuav dav cov phiaj xwm DNA ntau dua li ib lab zaug.Lub hauv paus ntsiab lus yog nyob rau hauv lub catalysis ntawm DNA polymerase, siv niam txiv strand DNA raws li ib tug template thiab tshwj xeeb primer raws li qhov pib taw tes rau extension.Nws yog replicated hauv vitro los ntawm cov kauj ruam xws li denaturation, annealing, thiab extension.Cov txheej txheem ntawm tus ntxhais strand DNA ntxiv rau niam txiv strand template DNA.

Tus txheej txheem PCR tau muab faib ua peb kauj ruam:

1.Denaturation: Siv qhov kub thiab txias los cais DNA ob txoj hlua.Lub hydrogen daim ntawv cog lus ntawm DNA ob strands yog tawg ntawm qhov kub thiab txias (93-98 ℃).

2.Annealing: Tom qab ob-stranded DNA sib cais, txo qhov kub kom cov primer tuaj yeem khi rau ib leeg-stranded DNA.

3.Extension: Lub DNA polymerase pib tsim cov khoom sib txuas ntxiv raws cov DNA strands los ntawm cov primers khi thaum qhov kub thiab txias.Thaum qhov txuas ntxiv ua tiav, lub voj voog tiav, thiab tus lej ntawm DNA tawg ob npaug

Reciprocating peb kauj ruam 25-35 zaug, tus naj npawb ntawm DNA tawg yuav nce exponentially.

Lub ingenuity ntawm PCR yog tias cov primers sib txawv tuaj yeem tsim los rau cov hom phiaj sib txawv, yog li lub hom phiaj cov noob tawg tuaj yeem nthuav dav hauv lub sijhawm luv luv.

Txog tam sim no, PCR tuaj yeem muab faib ua peb pawg, uas yog PCR zoo tib yam, fluorescent quantitative PCR thiab digital PCR.

Thawj tiam ntawm PCR zoo tib yam

Siv cov cuab yeej PCR zoo tib yam los nthuav tawm cov noob caj noob ces, thiab tom qab ntawd siv agarose gel electrophoresis txhawm rau txheeb xyuas cov khoom, tsuas yog kev soj ntsuam zoo tuaj yeem ua tiav.

Lub ntsiab tsis zoo ntawm thawj tiam PCR:

1.Nyob rau qhov tsis muaj kev nthuav dav thiab cov txiaj ntsig tsis zoo.

2.Qhov kev tshawb pom yuav siv sij hawm ntev thiab kev ua haujlwm yog cumbersome.

3.Tsuas yog qhov kev xeem zoo tuaj yeem ua tiav

Thib ob tiam Real-Time PCR

Real-Time PCR, tseem hu ua qPCR, siv cov fluorescent probes uas tuaj yeem qhia txog kev nce qib ntawm cov tshuaj tiv thaiv kab mob, thiab saib xyuas cov khoom sib txuas ntawm cov khoom lag luam los ntawm kev sib txuam ntawm cov teeb liab fluorescent, thiab txiav txim cov txiaj ntsig los ntawm fluorescence nkhaus.Nws tuaj yeem suav nrog kev pab ntawm Cq tus nqi thiab tus qauv nkhaus.

Vim tias qPCR thev naus laus zis tau ua tiav hauv lub kaw lus kaw, qhov tshwm sim ntawm kev sib kis tau raug txo, thiab cov teeb liab fluorescence tuaj yeem raug saib xyuas kom muaj nuj nqis, yog li nws yog qhov siv dav tshaj plaws hauv kev kho mob thiab tau dhau los ua cov thev naus laus zis hauv PCR.

Cov tshuaj fluorescent siv nyob rau hauv real-time fluorescent quantitative PCR yuav muab faib ua: TaqMan fluorescent sojntsuam, molecular beacons thiab fluorescent dye.

1) TaqMan fluorescent sojntsuam:

Thaum lub sij hawm PCR amplification, ib tug tshwj xeeb fluorescent sojntsuam yog ntxiv thaum ntxiv ib khub ntawm primer.Qhov kev sojntsuam yog ib qho oligonucleotide, thiab ob qhov kawg yog sau nrog cov neeg sau xov xwm fluorescent thiab pawg quencher fluorescent.

Thaum qhov kev sojntsuam tsis zoo, lub teeb liab fluorescent emitted los ntawm pawg neeg sau xov xwm yog absorbed los ntawm pawg quenching;Thaum lub sij hawm PCR amplification, lub 5′-3′ exonuclease kev ua ntawm Taq enzyme cleaves thiab degrades lub sojntsuam, ua tus reporter fluorescent pab pawg neeg thiab quencher Cov fluorescent pawg yog sib cais, kom lub fluorescence xyuas system tau txais lub fluorescence teeb liab, uas yog, txhua txhua lub sij hawm ib tug DNA strand fluorescent amplified, lub teeb liab yog ib tug amplified. tag nrho synchronized nrog tsim ntawm cov khoom PCR.

2) SYBR fluorescent zas xim:

Hauv PCR cov tshuaj tiv thaiv, qhov ntau tshaj ntawm SYBR fluorescent dye yog ntxiv.Tom qab SYBR fluorescent dye tsis yog tshwj xeeb koom ua ke rau hauv DNA ob txoj hlua, nws tawm lub teeb liab fluorescent.Lub SYBR dye molecule uas tsis muab tso rau hauv cov saw yuav tsis tawm ib qho teeb meem fluorescent, yog li ua kom lub teeb liab fluorescent Qhov nce hauv PCR cov khoom lag luam yog synchronized tag nrho nrog kev nce hauv PCR cov khoom.SYBR tsuas yog khi rau ob-stranded DNA, yog li ntawd lub melting nkhaus yuav siv tau los txiav txim seb cov tshuaj tiv thaiv PCR tshwj xeeb.

3) Molecular beacon:

Nws yog qia-voj ob-labeled oligonucleotide sojntsuam uas tsim cov qauv plaub hau ntawm txog 8 lub hauv paus ntawm 5 thiab 3 kawg.Cov nucleic acid sequences ntawm ob qho kawg yog ua ke ua ke, ua rau pawg fluorescent thiab pawg quenching nruj.Kaw, tsis muaj fluorescence yuav raug tsim.

Tom qab cov khoom PCR raug tsim, thaum lub sij hawm annealing txheej txheem, nruab nrab ib feem ntawm lub molecular beacon yog ua ke nrog ib tug tshwj xeeb DNA ib theem zuj zus, thiab cov fluorescent noob yog cais los ntawm lub quencher noob los tsim fluorescence.

Lub ntsiab tsis zoo ntawm ob tiam PCR:

Qhov kev xav tau tseem tsis muaj, thiab kev tshawb pom ntawm cov qauv luam tawm qis yog qhov tsis raug.

Muaj qhov cuam tshuam ntawm tus nqi keeb kwm yav dhau, thiab qhov tshwm sim yog raug cuam tshuam.

Thaum muaj PCR inhibitors hauv cov tshuaj tiv thaiv kab mob, qhov kev kuaj pom tau raug cuam tshuam.

Peb tiam digital PCR

Cov PCR (DigitalPCR, dPCR, Dig-PCR) suav cov lej luam ntawm lub hom phiaj sib lawv liag los ntawm qhov kawg-point detection, thiab tuaj yeem ua tiav qhov kev tshawb pom tseeb tsis tas siv cov kev tswj sab hauv thiab cov qauv nkhaus.

Digital PCR siv qhov kawg-point detection thiab tsis nyob ntawm tus nqi Ct (lub voj voog pib), yog li cov PCR cov tshuaj tiv thaiv tsis tshua muaj kev cuam tshuam los ntawm kev ua kom pom tseeb, thiab kev ua siab ntev rau PCR cov tshuaj tiv thaiv inhibitors tau txhim kho, muaj qhov tseeb thiab kev rov ua dua tshiab.

Vim yog cov yam ntxwv ntawm kev nkag siab zoo thiab qhov tseeb siab, nws tsis yooj yim cuam tshuam los ntawm PCR cov tshuaj tiv thaiv cov tshuaj tiv thaiv, thiab nws tuaj yeem ua tiav qhov tseeb tsis muaj cov khoom lag luam, uas tau dhau los ua kev tshawb fawb thiab thov hotspot.

Raws li cov ntaub ntawv sib txawv ntawm cov tshuaj tiv thaiv, nws tuaj yeem muab faib ua peb hom: microfluidic, nti thiab droplet systems.

1) Microfluidic digital PCR, mdPCR:

Raws li kev siv thev naus laus zis microfluidic, cov qauv DNA sib cais.Lub tshuab microfluidic tuaj yeem paub txog cov qauv nano-kho kho dua tshiab lossis tiam ntawm cov tee me me, tab sis cov tee dej xav tau ib txoj kev tshwj xeeb adsorption thiab tom qab ntawd ua ke nrog PCR cov tshuaj tiv thaiv.mdPCR tau maj mam tau txais los ntawm lwm txoj kev hloov.

2) Droplet-based digital PCR, ddPCR:

Siv cov dej-hauv-roj droplet technology los ua cov qauv rau hauv cov tee dej, thiab faib cov tshuaj tiv thaiv uas muaj cov nucleic acid molecules rau ntau txhiab tus nanoscale tee, txhua yam uas tsis muaj cov nucleic acid lub hom phiaj molecule yuav raug kuaj, los yog muaj ib mus rau ob peb nucleic acid lub hom phiaj molecules yuav tsum tau kuaj.

3) Chip-based digital PCR, cdPCR:

Siv cov txheej txheem ua kua dej sib xyaw ua ke los kos ntau lub microtubes thiab microcavities ntawm silicon wafers lossis quartz iav, thiab tswj cov dej ntws los ntawm cov kev tswj sib txawv, thiab faib cov qauv ua kua rau hauv nanometers ntawm qhov loj tib yam rau hauv cov tshuaj tiv thaiv qhov dej rau digital PCR Reaction kom ua tiav qhov ntau npaum li cas.

Lub ntsiab tsis zoo ntawm PCR tiam thib peb:

Cov khoom siv thiab cov tshuaj reagents yog kim.

Cov template zoo yuav tsum muaj siab.Yog tias tus qauv muaj ntau tshaj qhov microsystem kom muaj nuj nqis, nws yuav tsis tuaj yeem ua kom muaj nuj nqis, thiab yog tias nws tsawg dhau, qhov tseeb qhov ntau yuav raug txo.

Cov txiaj ntsig tsis tseeb kuj tuaj yeem tsim tawm thaum tsis muaj kev nthuav dav tshwj xeeb.