Qhov sib txawv tseem ceeb ntawm nick txhais lus thiab qhov kawg sau yog qhov kev txhais lus nick yog txheej txheem uas tsim cov ntawv sau DNA sojntsuam rau ntau yam kev sib xyaw ua ke thaum kawg txhaws yog cov txheej txheem uas tsim blunted fragments los ntawm kev ntxiv nucleotides rau hauv ib leeg-stranded overhangs.
Nick txhais lus thiab qhov kawg filling yog ob txoj kev siv hauv molecular biology. Nick txhais lus yog siv los sau cov ntawv sojntsuam rau hybridization txhawm rau txhawm rau txheeb xyuas cov nucleotide ib ntus. End filling yog siv los ua blunted fragments uas muaj nplaum kawg nrog ib leeg-stranded overhangs. Ob lub tswv yim muaj qhov tseem ceeb, thiab lawv niaj hnub ua hauv cov chaw tshawb fawb molecular.
Nick Translation yog dab tsi?
Nick txhais lus yog cov txheej txheem tseem ceeb siv los npaj cov ntawv sojntsuam rau ntau yam kev siv tshuaj lom neeg molecular xws li blotting, hauv situ hybridization, fluorescent hauv situ hybridization, thiab lwm yam. Nws yog ib txoj hauv kev hauv vitro ntawm DNA labelling. DNA sojntsuam yog siv los txheeb xyuas cov DNA lossis RNA ib ntus. Nrog kev pab los ntawm ib daim ntawv lo soj ntsuam, cov khoom tawg yooj yim tuaj yeem raug cim lossis pom los ntawm kev sib xyaw ntawm nucleic acid. Yog li ntawv sau npe probes tau npaj siv ntau yam txuj ci. Nick translation yog ib txoj hauv kev uas tsim cov ntawv soj ntsuam nrog kev pab ntawm DNase 1 thiab DNA polymerase 1 enzymes.
Daim duab 01: Kev txhais lus Nick
Nick translation process pib nrog DNase 1 enzyme kev ua. DNase 1 qhia nicks rau phosphate caj qaum ntawm ob-stranded DNA los ntawm cleaving phosphodiester bonds ntawm nucleotides. Thaum lub nick tsim, dawb 3′ OH pawg ntawm nucleotide yuav raug tsim, thiab DNA polymerase 1 enzyme yuav ua rau nws. 5′ rau 3′ exonuclease kev ua ntawm DNA polymerase 1 tshem tawm nucleotides ntawm lub nick mus rau 3′ kev taw qhia ntawm DNA strand.
Ib txhij, cov haujlwm polymerase ntawm DNA polymerase 1 enzyme ua haujlwm thiab ntxiv nucleotides los hloov cov nucleotides tshem tawm. Yog tias cov nucleotides tau sau npe, qhov hloov pauv yuav tshwm sim los ntawm cov ntawv sau npe nucleotides, thiab nws yuav cim DNA rau kev txheeb xyuas. Thaum kawg, cov ntawv sau tshiab DNA no tuaj yeem siv los ua kev sojntsuam hauv ntau yam kev sib xyaw ua ke.
End Filling yog dab tsi?
End filling yog cov txheej txheem siv hauv molecular biology los ua blunted fragments. Kev txwv kev zom zaub mov tsim cov khoom tawg nrog overhangs. Cov fragments no yuav tsis sib haum rau ligate rau hauv plasmid vectors. Vectors feem ntau yog blunted kom tso cai rau qhov tsis sib xws tau koom nrog. Yog li ntawd, fragments nrog overhangs yuav blunted los ntawm kev ntxiv cov nucleotides rau lub complementary strand siv lub overhang ua tus qauv rau polymerization. Cov txheej txheem no hu ua qhov kawg filling.
DNA polymerases xws li Klenow fragment ntawm DNA Polymerase I thiab T4 DNA Polymerase catalyze qhov kawg txhaws. Lawv ntxiv nucleotides rau hauv (5′ → 3′) thiab zom rov qab (3′ → 5′). Thaum cov nplaum nplaum tag lawm, lawv ua blunt, thiab lawv npaj txhij mus rau hauv ib lub vector.
Tsis tas li ntawd, qhov kawg filling tuaj yeem siv los sau cov DNA molecules. Txawm li cas los xij, nws tsuas yog siv tau los sau cov DNA molecules nrog cov nplaum kawg. Hauv kev sib piv rau kev txhais lus nick, qhov kawg sau yog txoj kev maj mam uas tsis tshua ua rau DNA tawg.
Dab tsi yog qhov zoo sib xws ntawm kev txhais lus Nick thiab Xaus Sau?
- Ob qho kev txhais lus nick thiab qhov kawg filling yog cov txheej txheem lom neeg lom neeg.
- Lawv tuaj yeem siv los sau npe soj ntsuam.
- Ob leeg nyob hauv vitro
Dab tsi yog qhov txawv ntawm Nick Txhais thiab Xaus Sau?
Nick txhais lus yog cov txheej txheem uas tsim cov ntawv sau npe rau kev sib xyaw. Nyob rau hauv sib piv, kawg filling yog ib tug txheej txheem uas tsim blunt fragments rau ligation rau hauv vectors. Yog li, qhov no yog qhov sib txawv tseem ceeb ntawm kev txhais lus nick thiab qhov kawg sau. Tsis tas li ntawd, Nick txhais lus yuav tsum tau siv 5'to 3' exonuclease kev ua thaum kawg filling tsis xav tau 5'to 3' exonuclease kev ua haujlwm.
Cov hauv qab no infographic tabulates qhov sib txawv ntawm nick txhais lus thiab xaus sau kom meej ntxiv.
Summary – Nick Translation vs End Filling
Nick txhais lus yog ib txoj hauv kev uas suav nrog radiolabeled nucleotides rau hauv DNA. Nws synthesizes labeled probes raws li cov dej num ntawm DNase 1 thiab E. coli DNA polymerase 1 enzymes. Thaum kawg filling, ntawm qhov tod tes, yog ib qho txheej txheem uas ua rau tawg tawg tawg. Thaum cov fragments muaj nplaum kawg (ib zaug-stranded overhangs), nws yog ib qhov tsim nyog los ua kom lawv blunted xaus thiaj li yuav ligate rau hauv vectors. Xaus filling ntxiv tau tshaj nucleotides thiab tsim blunt kawg fragments thiaj li ua rau lawv sib xws rau ligation. Yog li, qhov no qhia txog qhov sib txawv ntawm nick translation thiab xaus filling.
