Qhov txawv ntawm Cloning thiab Subcloning

2024 Tus sau: Alex Aldridge | [email protected]. Kawg hloov kho: 2024-01-17 23:34

Qhov Tseem Ceeb Qhov txawv - Cloning vs Subcloning

Cloning thiab Subcloning yog cov txheej txheem lom neeg molecular uas tsim cov caj ces zoo tib yam lossis cov kab mob uas muaj DNA lossis cov noob uas nyiam. Cloning yog ib qho txheej txheem uas cuam tshuam nrog kev nkag siab ntawm cov noob los yog DNA rau hauv ib qho vector, rov ua dua ntawm nws nyob rau hauv cov kab mob tus tswv, thiab tsim cov hlwb lossis cov kab mob uas yog cov ntawv luam ntawm cov caj ces. Subcloning yog ib qho txheej txheem uas cuam tshuam nrog kev nkag mus ntawm cov noob ntawm kev txaus siab, uas twb tau muab tso rau hauv ib qho vector, rau hauv cov vector thib ob, rov ua dua ntawm nws nyob rau hauv cov kab mob, thiab tsim cov genetic zoo tib yam cov ntawv luam ntawm cov hlwb lossis cov kab mob. Qhov tseem ceeb sib txawv ntawm cloning thiab subcloning yog hais tias, nyob rau hauv cloning, cov noob ntawm kev txaus siab, ib zaug ligated rau hauv ib tug vector, txuas ntxiv cov txheej txheem cloning thaum, nyob rau hauv lub subcloning, lub cloned noob ntawm kev txaus siab yog cais los ntawm niam txiv vector thiab ntxiv rau hauv. tus neeg txais vector thiab txuas ntxiv cov txheej txheem.

Dab tsi yog Cloning?

Cloning yog txheej txheem uas tsim cov kab mob sib xws lossis cov hlwb. Nyob rau hauv qhov xwm txheej, cloning tshwm sim nyob rau hauv txhais tau tias ntawm asexual reproduction. Thaum tsis muaj genetic recombination los yog hloov, cov ntxhais cell tau txais tib yam genetic pleev ntawm niam txiv. Cov kab mob Prokaryotic thiab eukaryotic tsim clones los ntawm binary fission, budding, mitosis, thiab lwm yam. Nyob rau hauv molecular biology, cloning genes los yog tej fragments ntawm DNA yog ib tug nrov txoj kev los kawm cov qauv thiab kev ua hauj lwm ntawm cov DNA tshwj xeeb seem.

Lub hom phiaj tseem ceeb ntawm molecular cloning yog ua kom ntau lab daim ntawv luam ntawm cov noob caj noob ces zoo tib yam lossis cov kab mob uas muaj cov DNA feem ntawm kev txaus siab (feem ntau yog cov noob). Nws tsim cov kab mob nrog cov ntawv luam ntawm caj ces tseeb ntawm lwm tus. Feem ntau, cov noob tshwj xeeb yog cloned hauv cov kev tshawb fawb molecular kom tau txais cov ntaub ntawv tsim nyog thiab kev ua haujlwm thiab rau DNA sequencing. Tsis tas li ntawd, rau kev tsim cov proteins tshwj xeeb lossis cov khoom lag luam loj, cloning yog dav siv.

Cloning txheej txheem

Cov kauj ruam yooj yim ntawm cov txheej txheem cloning yog raws li hauv qab no.

1. Kev txheeb xyuas thiab cais cov noob ntawm kev txaus siab. (Kev nthuav dav ntawm cov noob ntawm kev txaus siab los ntawm PCR).
2. Kev txwv kev zom zaub mov ntawm cov noob paj hlwb (Kev txwv endonuclease txiav cov noob).
3. Kev txwv kev zom zaub mov ntawm vector DNA. (Vector DNA kuj raug txiav siv tib yam kev txwv endonuclease).
4. Kev tso cov noob rau hauv vector thiab tsim cov molecule recombinant.
5. Kev hloov pauv ntawm cov vector rov ua dua rau hauv cov kab mob hauv tsev.
6. Kev cais tawm thiab txheeb xyuas cov kab mob hloov pauv (plasmid vector yuav tsum muaj cov noob xaiv tau, feem ntau yog cov tshuaj tiv thaiv kab mob rau tshuaj ntsuam cov kab mob hloov pauv).
7. Recombinant gene qhia hauv tus tswv.

Figure_01: Cloning Procedure

Subcloning yog dab tsi?

Subcloning yog txheej txheem ntawm kev txav cov noob ntawm kev txaus siab los ntawm ib tus vector mus rau lwm tus vector kom pom qhov kev qhia ntawm cov noob kom tau txais qhov kev xav tau ntawm cov noob. Hauv txoj kev no, ob lub vectors koom nrog; yog, niam txiv vector thiab destination vector. Cloned inserts yog tsiv dua mus rau hauv ib tug thib ob vector nyob rau hauv subcloning. Lub hom phiaj ntawm kev hloov cov noob los ntawm thawj vector mus rau qhov thib ob vector yog kom tau ib yam dab tsi uas tsis tuaj yeem ua los ntawm thawj vector lossis cais cov noob ntxiv nyob rau hauv cov kab ke uas twb muaj lawm ntawm DNA thiab qhia nws ib leeg. Kev txwv enzymes yog siv rau hauv cov txheej txheem no thaum pib.

txheej txheem subcloning

Cov kauj ruam yooj yim ntawm subcloning yog raws li hauv qab no.

1. Nrog kev pab los ntawm kev txwv endonucleases, kev sib cais ntawm DNA ntawm kev txaus siab rau tus neeg pub plasmid (niam txiv vector).
2. Kev nthuav dav ntawm DNA ntawm kev txaus siab siv PCR.
3. Kev huv ntawm cov khoom PCR (DNA ntawm kev txaus siab) los ntawm gel electrophoresis.
4. Qhib tus neeg txais plasmid los ntawm tib yam kev txwv endonucleases siv los cais DNA ntawm kev txaus siab rau niam txiv plasmid.
5. Ligation ntawm DNA ntawm kev txaus siab (gene) rau hauv tus neeg tau txais plasmid los tsim cov subcloned plasmid.
6. Kev hloov pauv ntawm subcloned vector rau hauv cov kab mob muaj peev xwm.
7. Kev tshuaj ntsuam ntawm cov hlwb hloov pauv.
8. Kev huv ntawm cov plasmid DNA thiab siv rau DNA sequencing lossis qhia cov noob kom tau cov khoom xav tau.

Subcloning yog ua tiav nyob rau lub sijhawm ntawm kev cais ib tus noob los ntawm pawg cloned ntawm cov noob lossis thaum cov noob ntawm kev txaus siab yuav tsum tau hloov mus rau hauv cov txiaj ntsig plasmid kom pom qhov tseeb ntawm cov noob ntawm kev txaus siab.

Figure_02: Subcloning Procedure

Qhov txawv ntawm Cloning thiab Subcloning yog dab tsi?

Cloning vs Subcloning
Cloning yog txheej txheem uas tsim cov kab mob sib xws lossis cov hlwb.	Subcloning yog txheej txheem ntawm kev txav cov noob ntawm kev txaus siab los ntawm ib tus vector mus rau lwm tus vector kom pom qhov kev qhia ntawm cov noob kom tau txais cov txiaj ntsig xav tau ntawm cov noob.
txheej txheem
Cais DNA ntawm kev txaus siab los ntawm lub cev thiab muab tso rau hauv vector ib zaug thiab cloned	Tam sim no cloned DNA yog cais los ntawm thawj vector thiab muab tso rau hauv ib tug thib ob vector thiab cloned.
Insert Movement via Vectors
Tsis txav cov ntxig (DNA ntawm kev txaus siab) ntawm ib lub vector mus rau lwm tus vector.	Txav cov ntxig los ntawm niam txiv vector mus rau qhov chaw vector.

Summary – Cloning vs Subcloning

Cloning tsim cov noob caj ces zoo ib yam lossis cov kab mob nrog cov noob los yog DNA ntawm kev txaus siab. Nws tshwm sim los ntawm kev sib cais thiab tso rau ntawm DNA ntawm kev txaus siab rau hauv vector thiab kev nthuav qhia nyob rau hauv tus kab mob tus tswv. Subcloning qhia cov kauj ruam zoo sib xws nrog cloning. Txawm li cas los xij, hauv subcloning, twb tau cloned DNA fragment (gene ntawm kev txaus siab) yog muab tso rau hauv ib tug vector thiab hloov mus rau hauv tus tswv tsev kab mob. Qhov ntawd yog qhov sib txawv tseem ceeb ntawm cloning thiab subcloning.