Qhov Tseem Ceeb Qhov txawv - Blunt vs Sticky End Ligation
Kev txwv endonucleases yog cov enzymes tshwj xeeb uas txiav ob-stranded DNA (dsDNA). Lawv tseem hu ua molecular txiab hauv Molecular biology. Kev txwv enzymes muaj peev xwm paub txog cov ntu luv luv ntawm dsDNA lub npe hu ua qhov chaw lees paub thiab cleave phosphodiester thiab hydrogen bonds qhib ob lub strands. Raws li qhov tshwm sim ntawm cleavage los ntawm cov enzymes no, DNA tawg yog tsim nrog ntau hom xaus xws li nplaum kawg thiab blunt kawg. DNA ligase yog ib qho enzyme siv nyob rau hauv Molecular biology los koom nrog ob kab uas nyob ib sab ntawm DNA los ntawm kev tsim cov ntawv cog lus tshiab. Cov kauj ruam yog hu ua ligation thiab raws li hom DNA kawg ligated, lawv tuaj yeem raug hu ua blunt kawg ligation thiab nplaum kawg ligation. Qhov sib txawv tseem ceeb ntawm blunt thiab nplaum kawg ligation yog tias blunt kawg ligation tshwm sim ntawm DNA fragments uas muaj ob blunt kawg whereas nplaum kawg ligation tshwm sim ntawm 5' thiab 3' overhangs. Piv rau blunt kawg ligation, nplaum kawg ligation yog ntau npaum thiab ruaj khov.
Dab tsi yog Blunt End Ligation?
Qee qhov kev txwv endonucleases tuaj yeem txiav cov DNA ntawm cov hauv paus rov qab thiab tsim cov DNA tsis zoo. Cov enzymes no hu ua blunt kawg cutters; lawv cleave ncaj nraim mus rau nruab nrab ntawm qhov chaw txwv tsis pub tawm ib qho stranded overhanging bases. Blunt kawg tseem hu ua non-overhanging xaus vim lawv tsis muaj 3' thiab 5' overhanging bases ntawm qhov kawg. Ob lub strands txiav tawm los ntawm lub hauv paus khub hauv blunt kawg. Feem ntau blunt kawg txiav enzymes yog EcoRV HaeIII, AluI thiab SmaI.
Blunt kawg ligation yog koom nrog ntawm ob qhov kawg. Nws tsis yog ib qho ligation ntawm protruding bases. Qhov kev ligation no tsis tshua muaj txiaj ntsig ntau dua li cov nplaum kawg ligation. Txawm li cas los xij, qee lub sijhawm, blunt kawg ligation ua tau zoo dua li cov nplaum kawg ligation, tshwj xeeb tshaj yog thaum ligating PCR cov khoom. PCR cov khoom yog ib txwm tsim nrog blunt kawg. Blunt kawg ligation tsis tas yuav xaus rau hauv DNA rau ligation.
Daim duab 01: Blunt kawg tsim los ntawm Eco RV enzyme
Dab tsi yog Qhov Ncauj Kawg Ligation?
Qee qhov kev txwv endonucleases tuaj yeem txiav dsDNA, tawm hauv ib qho ntawm ib leeg-stranded DNA thaum kawg. Cov xaus no yog hu ua nplaum los yog overhanging xaus. Cov nplaum kawg ligation tshwm sim ntawm ob qhov DNA seem uas muaj qhov sib txuam overhangs vim cov nplaum kawg yog muaj nrog cov hauv paus tsis sib xws thiab xav tau cov hauv paus ntxiv los tsim cov ntawv cog lus. Yog li ntawd, nws yog ib qho tsim nyog yuav tsum siv tib qhov kev txwv enzyme rau ob qho tib si ntawm DNA los tsim qhov sib txuam ligating fragments.
Cov nplaum kawg ligation yog qhov ua tau zoo dua thiab feem ntau xav tau hauv cov txheej txheem cloning. Muaj ntau qhov txwv enzymes uas tsim cov nplaum kawg. Lawv yog EcoRI, BamHI, HindIII thiab lwm yam.
Daim duab 02: Nqus qhov kawg tsim los ntawm Eco RI enzyme
Qhov txawv ntawm Blunt thiab Sticky End Ligation yog dab tsi?
Blunt vs Sticky End Ligation |
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| Blunt kawg ligation tshwm sim ntawm ob qhov kawg DNA tawg. | Nco kawg ligation tshwm sim ntawm ob qhov sib txuam DNA tawg nrog cov nplaum kawg. |
| Enzymes | |
| Blunt kawg cutters tsim blunt kawg. | Npaj kawg cutters ua nplaum los yog cohesive kawg. |
| Yuav tsum muaj kev sib tw kawg | |
| Nws tsis tas yuav muaj qhov sib luag lossis cov hauv paus ntxiv. | Nws xav tau cov hauv paus ntxiv ntawm qhov kawg los tsim cov hauv paus khub. |
| Efficiency | |
| Nws ua haujlwm tsawg dua li nplaum kawg ligation | Nws ua tau zoo dua li qhov kawg ligation. |
Summary – Blunt vs Sticky End Ligation
Txoj kev txwv endonucleases tuaj yeem tshem tawm dsDNA thiab tsim cov DNA tawg nrog qhov sib txawv. Lawv paub txog cov kab ke tshwj xeeb thiab txwv DNA tsim cov nplaum thiab blunt kawg. Nplaum kawg muaj unpaired hauv paus ntawm qhov kawg ntawm fragments. Blunt kawg yog tsim los ntawm ib tug ncaj cleavage thiab lawv muaj lub hauv paus khub ntawm qhov kawg. Cov nplaum kawg ligation yuav tsum muaj ob qhov sib ntxiv ntawm DNA ib leeg. Blunt kawg ligation tshwm sim ntawm ob qho tag nrho blunt kawg fragments. Qhov no yog qhov txawv ntawm blunt thiab nplaum ligation.
