Qhov cuam tshuam txog cov yam hauv PCR kev

Thaum lub sijhawm pccr kev tawm tsam, qee qhov cuam tshuam qee yam feem ntau ntsib.
Vim yog qhov kev rhiab siab heev ntawm PCR, paug tau txiav txim siab yog ib qho tseem ceeb tshaj plaws cuam tshuam rau cov txiaj ntsig pccries thiab tau ua cov txiaj ntsig tsis tseeb.
Sib luag tseem ceeb yog ntau qhov chaw uas ua rau cov txiaj ntsig tsis muaj tseeb. Yog tias ib lossis ntau qhov tseem ceeb ntawm PCR sib xyaw lossis lub zog kev tiv thaiv nws tus kheej yog kev cuam tshuam lossis cuam tshuam nrog, cov kev kuaj mob tuaj yeem cuam tshuam. Qhov no tuaj yeem ua rau txo qis kev ua haujlwm thiab txawm tias tsis zoo cov txiaj ntsig.
Ntxiv nrog rau kev cuam tshuam, ploj ntawm phiaj nucleic acid kev ncaj ncees yuav tshwm sim vim kev xa khoom thiab / lossis kev cia khoom ua ntej npaj. Tshwj xeeb, kev ntsuas kub tsis txaus los yog kev cia kom txaus ua rau kev puas tsuaj hlwb thiab nucleic acids. Cell thiab cov nqaij kho thiab kho cov phev thiab cov tshuaj tua kab uas paub zoo ua rau muaj teeb meem DNA thiab muaj teeb meem 1 thiab 2). Hauv cov xwm txheej no, txawm tias kev sib cais thiab kev ua kom huv siab yuav tsis pab.

Daim duab 1 | Cov nyhuv ntawm immobilization ntawm DN SNA Kev Ncaj Ncees
Agarose gel electrophoresis qhia tau tias qhov zoo ntawm DNA cais los ntawm cov tshooj lus qhia ntawm autopsies variedly. DNA ntawm txawv nruab nrab tawg qhov ntev tau tam sim no nyob rau hauv cov kev rho tawm nyob ntawm txoj kev txhim kho. DNA tau khaws cia tsuas yog thaum tsau ntsuas cov qauv khov thiab hauv kev ua tsis ncaj ncees. Kev siv ntawm cov acidic didic bouin txhim kho lossis unbuffered, formic acid-muaj cov formalin ua rau DNA tseem ceeb. Qhov seem seem yog cov tawg heev.
Ntawm sab laug, qhov ntev ntawm cov tawg yog qhia nyob rau hauv kilobase khub (KBP)

Daim duab 2 | Poob ntawm kev ncaj ncees ntawm nucleic acid phiaj
(a) Ib qho 3 '-5' sib txawv ntawm ob qho tib si strands yuav ua rau muaj kev so hauv lub hom phiaj DNA. synthesis ntawm DNA tseem yuav tshwm sim ntawm cov thooj me me. Txawm li cas los xij, yog tias lub vev xaib prevealing yog ploj ntawm DNA fribment, tsuas yog linear amplification tshwm sim. Nyob rau hauv cov txiaj ntsig zoo tshaj plaws, cov tawg hniav yuav resaturate sib, tab sis cov yields yuav muaj cov qib me thiab hauv qab.
(b) poob ntawm cov hauv paus, feem ntau yog vim muaj nqis thiab thymidine Dimer tsim, ua rau txo qis ntawm H-Bonds thiab txo qis hauv tm. Thaum lub sij hawm elongated sov sov theem, cov primers yuav yaj ntawm matrix dNA thiab yuav tsis muaj anneal txawm tias tsis muaj kev nruj me ntsis.
(c) Cov thoo uas nyob ib sab thoo uas ua ib tt Dimer.
Lwm qhov teeb meem tshwm sim uas feem ntau tshwm sim hauv molecular kev kuaj mob yog qhov tsawg dua-zoo tshaj tawm ntawm lub hom phiaj nucleic acids piv rau phenol-chloroform extraction. Hauv cov xwm txheej hnyav, qhov no tuaj yeem cuam tshuam nrog cov tsis tseeb negatives. Ntau lub sijhawm tuaj yeem txuag tau los ntawm cov lysis boiling lossis enzymatic zom cov khib nyiab ntawm tes, tab sis cov qauv no feem ntau ua rau muaj kev nkag siab zoo ntawm pensitivity vim tsis muaj nucleic acid tso tawm.

Inhibition ntawm polymerase kev ua thaum lub caij

Feem ntau, Inhibition yog siv raws li lub tswv yim ntim cov tswv yim los piav txog txhua yam uas ua rau suboptimal pcr kev. Hauv kev txiav txim siab nruj me ntsis biochemical, inhibition yog txwv rau kev ua si ntawm cov enzyme, piv txwv li cov khoom siv ntawm DNA polymerase lossis nws cov cofactle (piv txwv li ntawm mg2 + rau Taq DNA polymerase).
Cheebtsam hauv cov qauv lossis ntau yam buffers thiab rho tawm muaj cov reagents tuaj yeem tiv thaiv polymerase (piv txwv li ua kom poob qis lossis tsis tseeb pcr kev.
Txawm li cas los xij, ntau yam kev sib cuam tshuam ntawm cov tshuaj tivthaiv thiab cov hom phiaj-muaj nucleic acids kuj tseem raug tsim raws li 'pcr inibitors'. Thaum lub cell yog cuam tshuam los ntawm kev sib cais thiab nucleic acid tawm, cuam tshuam nrog cov qauv thiab cov theem ntawm cov kev daws teeb meem thiab cov khoom lag luam. Piv txwv li, 'tsis txaus siab' tuaj yeem khi ib leeg- los ntawm kev sib cais thiab kev ua kom muaj kev sib cais los ntawm kev sib cais ntawm cov phiaj xwm ntawm pcr kev.
Feem ntau, pCreferors yog tam sim no nyob rau hauv feem ntau lub cev kua thiab reagents siv rau cov zis kuaj mob (organic, rog) thiab cov khoom siv hauv ib puag ncig (phenols , Cov Hlau Hnyav)

Ntau ntau cov pcr neeg ua yeeb ncuab tuaj yeem nrhiav tau hauv cov kab mob thiab cov kab mob hlwb eukaryotic, tsis muaj hom phiaj DNA, DNA-broading matricolecules ntawm cov nqaij matrices thiab cov khoom siv sim xws li hnab looj tes thiab yas. Kev ua kom huv ntawm nucleic acids thaum lossis tom qab extraction yog txoj kev nyiam dua rau kev tshem tawm pcR inhibitors.
Niaj hnub no, ntau cov khoom siv cov khoom siv tawm suab tuaj yeem hloov ntau hom kev cai, tab sis 100% rov qab thiab / lossis cov hom phiaj tsis tau tiav. Muaj peev xwm inibitors tseem yuav muaj nyob rau hauv lub suab muaj nucleic acids lossis tej zaum yuav tau ua tiav. Cov tswv yim sib txawv nyob rau txo qhov kev cuam tshuam ntawm inhibitors. Kev xaiv ntawm daim ntawv tsim nyog polymerase tuaj yeem muaj kev cuam tshuam tseem ceeb ntawm kev ua kom muaj kev sib raug zoo. Lwm txoj hauv kev los txo pCR inhibition yog nce polymerase lossis thov cov khoom ntxiv xws li BSA.
Kev txwv ntawm PCR cov kev cai lij choj tuaj yeem pom tau los ntawm kev siv cov txheej txheem sab hauv zoo (IPC).
Kev saib xyuas yuav tsum tau muab tshem tawm txhua qhov kev daws teeb meem thiab lwm yam kev daws teeb meem, etrop, ntawm nucleic acid cais tawm los ntawm ib kauj ruam los ntxuav. Nyob ntawm lawv txoj kev xav, lawv yuav qhib lossis tiv thaiv PC.