Kev cuam tshuam cuam tshuam hauv PCR cov tshuaj tiv thaiv

Thaum lub sijhawm PCR cov tshuaj tiv thaiv, qee qhov cuam tshuam cuam tshuam feem ntau ntsib.
Vim yog PCR rhiab heev, kev sib kis tau raug suav tias yog ib qho tseem ceeb tshaj plaws cuam tshuam rau PCR cov txiaj ntsig thiab tuaj yeem tsim cov txiaj ntsig tsis zoo.
Qhov tseem ceeb sib npaug yog ntau qhov chaw uas ua rau cov txiaj ntsig tsis tseeb-tsis zoo. Yog tias ib lossis ntau qhov tseem ceeb ntawm PCR sib xyaw lossis cov tshuaj tiv thaiv amplification nws tus kheej raug cuam tshuam los yog cuam tshuam nrog, qhov kev kuaj mob tuaj yeem cuam tshuam. Qhov no tuaj yeem ua rau txo qis kev ua haujlwm thiab txawm tias cov txiaj ntsig tsis zoo.
Ntxiv rau qhov inhibition, poob ntawm lub hom phiaj nucleic acid kev ncaj ncees tuaj yeem tshwm sim vim kev xa khoom thiab / lossis kev cia khoom ua ntej kev npaj ua qauv. Tshwj xeeb, qhov kub thiab txias los yog cia tsis txaus yuav ua rau puas hlwb thiab nucleic acids. Cell thiab cov ntaub so ntswg fixation thiab paraffin embedding yog paub zoo tias ua rau DNA fragmentation thiab muaj teeb meem tsis tu ncua (saib daim duab 1 thiab 2). Hauv cov xwm txheej no, txawm tias qhov kev sib cais zoo tshaj plaws thiab kev huv huv yuav tsis pab.

Daim duab 1 | Cov nyhuv ntawm immobilization ntawm DNA kev ncaj ncees
Agarose gel electrophoresis qhia tau hais tias qhov zoo ntawm DNA cais los ntawm paraffin seem ntawm kev txiav txim siab txawv txav. DNA ntawm qhov sib txawv nruab nrab qhov ntev ntawm qhov sib txawv tau tshwm sim nyob rau hauv cov extracts nyob ntawm txoj kev fixation. DNA tau khaws cia tsuas yog thaum kho nyob rau hauv cov qauv khov nab kuab thiab nyob rau hauv buffered nruab nrab formalin. Kev siv cov tshuaj muaj zog acidic Bouin fixative los yog unbuffered, formic acid-muaj formalin ua rau muaj qhov tsis zoo ntawm DNA. Cov seem seem yog fragmented heev.
Ntawm sab laug, qhov ntev ntawm cov khoom tawg yog qhia hauv kilobase khub (kbp)

Daim duab 2 | Kev poob ntawm kev ncaj ncees ntawm nucleic acid lub hom phiaj
(a) Qhov sib txawv ntawm 3'-5' ntawm ob txoj hlua yuav ua rau tawg ntawm lub hom phiaj DNA. synthesis ntawm DNA tseem yuav tshwm sim ntawm qhov me me. Txawm li cas los xij, yog tias qhov chaw primer annealing ploj lawm ntawm DNA tawg, tsuas yog linear amplification tshwm sim. Nyob rau hauv cov ntaub ntawv zoo tshaj plaws, cov khoom tawg tuaj yeem rov ua dua ib leeg, tab sis cov txiaj ntsig yuav me me thiab qis dua qib kev tshawb pom.
(b) Kev poob ntawm cov hauv paus, feem ntau yog vim depurination thiab thymidine dimer tsim, ua rau txo qis ntawm H-bonds thiab txo Tm. Thaum lub sij hawm elongated warming theem, cov primers yuav yaj tawm ntawm matrix DNA thiab yuav tsis anneal txawm nyob rau hauv tsawg zog tej yam kev mob.
(c) Nyob ib sab thymine bases tsim TT dimer.
Lwm qhov teeb meem tshwm sim uas feem ntau tshwm sim hauv kev kuaj mob molecular yog qhov tsawg tshaj li qhov zoo tshaj tawm ntawm lub hom phiaj nucleic acids piv rau phenol-chloroform extraction. Hauv qhov xwm txheej hnyav, qhov no tuaj yeem cuam tshuam nrog kev tsis zoo cuav. Ntau lub sij hawm tuaj yeem txuag tau los ntawm kev kub nyhiab los yog kev zom zom ntawm cov khib nyiab ntawm tes, tab sis txoj kev no feem ntau ua rau PCR rhiab heev vim tsis txaus nucleic acid tso tawm.

Inhibition ntawm polymerase kev ua si thaum lub sij hawm amplification

Feem ntau, inhibition yog siv los ua lub ntsiab lus ntim los piav txog txhua yam uas ua rau cov txiaj ntsig PCR tsis zoo. Hauv kev txiav txim siab biochemical nruj, inhibition tsuas yog txwv rau kev ua ntawm cov enzyme, piv txwv li, nws txo lossis tiv thaiv kev hloov pauv ntawm cov khoom siv los ntawm kev cuam tshuam nrog lub chaw nquag ntawm DNA polymerase lossis nws cov cofactor (xws li Mg2+ rau Taq DNA polymerase).
Cheebtsam hauv cov qauv lossis ntau yam buffers thiab cov extracts uas muaj reagents tuaj yeem cuam tshuam ncaj qha rau cov enzyme lossis ntxiab nws cov cofactors (xws li EDTA), yog li inactivating cov polymerase thiab nyob rau hauv lem ua rau txo los yog cuav tsis zoo PCR.
Txawm li cas los xij, ntau qhov kev sib cuam tshuam ntawm cov tshuaj tiv thaiv thiab lub hom phiaj muaj nucleic acids kuj raug xaiv los ua 'PCR inhibitors'. Thaum qhov kev ncaj ncees ntawm tes raug cuam tshuam los ntawm kev sib cais thiab cov nucleic acid raug tso tawm, kev sib cuam tshuam ntawm tus qauv thiab nws cov tshuaj nyob ib puag ncig thiab cov theem khoom tuaj yeem tshwm sim. Piv txwv li, 'scavengers' tuaj yeem khi ib leeg- lossis ob-kab DNA los ntawm kev tsis sib haum xeeb thiab cuam tshuam nrog kev sib cais thiab kev huv los ntawm kev txo tus naj npawb ntawm cov hom phiaj uas nws thiaj li ncav cuag PCR cov tshuaj tiv thaiv.
Feem ntau, PCR inhibitors muaj nyob rau hauv feem ntau cov kua hauv lub cev thiab cov tshuaj reagents siv rau kev kuaj mob kuaj mob (urea hauv cov zis, hemoglobin thiab heparin hauv cov ntshav), cov khoom noj khoom haus (organic Cheebtsam, glycogen, rog, Ca2+ ions) thiab cov khoom hauv ib puag ncig (phenols). , hnyav hlau)

Ntau cov PCR inhibitors tuaj yeem pom muaj nyob hauv cov kab mob thiab cov hlwb eukaryotic, tsis yog hom phiaj DNA, DNA-binding macromolecules ntawm cov ntaub so ntswg matrices thiab cov khoom siv kuaj xws li hnab looj tes thiab yas. Purification ntawm nucleic acids thaum lub sij hawm los yog tom qab rho tawm yog txoj kev nyiam rau tshem tawm PCR inhibitors.
Niaj hnub no, ntau yam khoom siv automated extraction tuaj yeem hloov tau ntau phau ntawv raws tu qauv, tab sis 100% rov qab thiab / lossis kev ua kom huv ntawm lub hom phiaj tsis tau ua tiav. Muaj peev xwm inhibitors tseem tuaj yeem muaj nyob rau hauv cov purified nucleic acids los yog twb tau siv lawm. Muaj cov tswv yim sib txawv los txo qhov cuam tshuam ntawm inhibitors. Kev xaiv ntawm cov polymerase uas tsim nyog tuaj yeem muaj kev cuam tshuam loj rau kev ua haujlwm inhibitor. Lwm cov kev pov thawj los txo cov PCR inhibition yog nce polymerase concentration lossis siv cov tshuaj ntxiv xws li BSA.
Inhibition ntawm PCR cov tshuaj tiv thaiv tuaj yeem pom tau los ntawm kev siv cov txheej txheem kev tswj hwm sab hauv (IPC).
Kev saib xyuas yuav tsum tau ua kom tshem tawm tag nrho cov reagents thiab lwm yam kev daws teeb meem hauv cov khoom siv rho tawm, xws li ethanol, EDTA, CETAB, LiCl, GuSCN, SDS, isopropanol thiab phenol, los ntawm nucleic acid cais los ntawm kev ntxuav kom huv. Nyob ntawm lawv cov concentration, lawv tuaj yeem qhib lossis inhibit PCR.