Qhov Tseem Ceeb Qhov txawv - Maxam Gilbert vs Sanger Sequencing
Nucleotides yog cov qauv tsim thiab tsim cov blocks ntawm DNA. DNA molecule yog tsim los ntawm cov saw hlau polynucleotide. Muaj plaub qhov sib txawv nucleotides pom hauv DNA. Cov nucleotides no yog tsim los ntawm plaub qhov sib txawv nitrogenous bases hu ua A (adenine), G (guanine), C (cytosine), T (thymine). Qhov kev txiav txim ntawm nucleotides hauv DNA molecule muaj qhov tseem ceeb vim nws encodes cov ntaub ntawv tseem ceeb ntawm caj ces rau cov kab mob loj hlob thiab kev loj hlob. DNA sequencing yog hais txog cov txheej txheem uas txiav txim siab qhov tseeb nucleotide sib lawv liag ntawm DNA. Muaj cov txheej txheem DNA sib txawv. Maxam Gilbert sequencing thiab Sanger DNA sequencing yog ob txoj kev ntawm DNA sequencing uas yog thawj tiam sequencing. Maxam Gilbert sequencing cov txheej txheem txiav txim siab lub hauv paus ua ntu zus los ntawm cov tshuaj cleaving 5 'kawg sau lo lus DNA fragments nyiam ntawm txhua ntawm plaub nucleotides thiab gel electrophoresis. Sanger sequencing txheej txheem txiav txim siab cov nucleotide sib lawv liag los ntawm kev sib txuas ib leeg-stranded DNA siv DNA polymerase thiab dideoxynucleotides thiab gel electrophoresis. Qhov no yog qhov sib txawv tseem ceeb ntawm Maxam Gilbert thiab Sanger Sequencing.
Maxam Gilbert Sequencing yog dab tsi?
Maxam Gilbert sequencing, tseem hu ua chemical sequencing method, yog cov txheej txheem uas tau tsim los txiav txim siab qhov kev txiav txim ntawm nucleotides hauv DNA. Txoj kev no tau qhia los ntawm W alter Gilbert thiab Alan Maxam hauv xyoo 1976 thiab tau los ua neeg nyiam vim nws tuaj yeem ua ncaj qha nrog DNA purified. Maxam Gilbert txoj kev belongs rau thawj tiam ntawm DNA sequencing, thiab nws yog thawj sequencing txoj kev siv dav los ntawm cov kws tshawb fawb.
Lub hauv paus ntsiab lus ntawm txoj kev no yog nrog kev txwv ntawm qhov kawg-labeled DNA fragments ntawm cov hauv paus tshwj xeeb los ntawm cov tshuaj hauv paus tshwj xeeb thiab cov xwm txheej thiab kev sib cais ntawm cov ntawv sau los ntawm electrophoresis raws li qhia hauv daim duab 01. Fragments raug cais raws li rau lawv qhov ntau thiab tsawg ntawm gel. Txij li cov khoom tawg tau sau tseg, cov kab ke ntawm DNA molecule tuaj yeem txiav tau yooj yim.
Maxam gilbert txoj kev siv cov tshuaj tshwj xeeb los ua txhaum DNA ntawm cov hauv paus tshwj xeeb. Ob hom tshuaj muaj npe hu ua dimethyl sulphate thiab hydrazine chemicals yog siv los xaiv los tua purines thiab pyrimidines, ntsig txog.
Maxam Gilbert sequencing method yog ua los ntawm ob peb kauj ruam raws li hauv qab no.
- Kev huv ntawm DNA ib ntus siv kev txwv endonucleases
- Sau npe ntawm qhov kawg ntawm DNA tawg los ntawm kev ntxiv cov phosphates radioactive
- Kev huv ntawm cov ntawv sau los ntawm cov khoom tsis muaj npe los ntawm gel electrophoresis
- Sib cais ntawm qhov kawg-labeled DNA rau hauv plaub lub raj thiab kho nrog cov tshuaj tshwj xeeb cais
- Electrophoresis ntawm cov ntsiab lus ntawm txhua lub raj ntawm cov kab sib cais ntawm cov gel thiab cov khoom sib cais raws li lawv qhov ntev.
- Ntseeg ntawm cov fragments by autoradiograph.
Daim duab 01: Maxam Gilbert Sequencing
Sanger Sequencing yog dab tsi?
Sanger Sequencing yog ib txoj hauv kev tsim los ntawm Frederick Sanger thiab nws cov npoj yaig hauv xyoo 1977 los txiav txim seb lub hauv paus sib lawv liag ntawm DNA tawg. Nws tseem hu ua chain termination sequencing lossis Dideoxy sequencing method. Txoj kev no ua haujlwm ntawm lub hauv paus ntsiab lus ntawm kev xaiv kev sib koom ua ke ntawm cov saw hlau txiav tawm dideoxynucleotides (ddNTPs) xws li ddGTP, ddCTP, ddATP thiab ddTTP los ntawm DNA polymerase thaum lub sij hawm synthesis ntawm ib leeg stranded DNA los txiav strand tsim. Dideoxynucleotides tsis muaj 3 'OH pawg rau kev tsim cov phosphodiester bonds nrog nucleotide nyob ib sab. Li no, txoj kev tsim strand nres ib zaug ib ddNTP tau muab tso rau hauv kev tsim tshiab strand thaum lub sij hawm sanger sequencing.
Nyob rau hauv txoj kev no, plaub cais DNA synthesis cov tshuaj tiv thaiv (PCR) tau ua nyob rau hauv plaub lub raj cais nrog ib hom ddNTP. Lwm qhov yuav tsum tau muab rau cov raj rau PCR suav nrog primers, dNTPs, Taq polymerase, cov xwm txheej tshwj xeeb, thiab lwm yam. Plaub qhov kev sib cais yog ua nyob rau hauv plaub lub raj nrog plaub sib tov. Tom qab PCR cov tshuaj tiv thaiv, qhov ua rau DNA tawg yog kub denatured thiab sib cais los ntawm gel electrophoresis. Tom qab ntawd cov khoom tawg yog pom los ntawm kev siv cov ntawv sau (xov tooj cua lossis fluorescent) primer lossis dNTPs raws li qhia hauv daim duab 02.
Daim duab 02: Sanger Sequencing
Qhov txawv ntawm Maxam Gilbert thiab Sanger Sequencing yog dab tsi?
Maxam Gilbert vs Sanger Sequencing |
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| Maxam Gilbert sequencing yog thawj txheej txheem tsim rau DNA sequencing. | Sanger sequencing method tau qhia tom qab Maxam Gilbert sequencing method. |
| Txoj kev no tsis tshua siv. | Sanger sequencing yog ib txwm siv los ua sequencing. |
| Kev siv tshuaj phom sij | |
| Nws siv tshuaj lom neeg. | Kev siv tshuaj lom neeg raug txwv tsis pub piv rau Maxam Gilbert txoj kev. |
| Labeling for Detection | |
| Txoj kev no siv cov xov tooj cua P32 rau sau npe qhov kawg ntawm DNA tawg. | Sanger sequencing siv xov tooj cua lossis fluorescently sau ddNTPs. |
Summary – Maxam Gilbert vs Sanger Sequencing
Maxam Gilbert thiab Sanger sequencing yog ob hom DNA sequencing cov txheej txheem los ntawm thawj tiam DNA sequencing. Maxam Gilbert sequencing yog thawj txoj kev qhia txog DNA sequencing hauv xyoo 1976, thiab nws tau ua los ntawm kev rhuav tshem qhov kawg sau npe DNA tawg los ntawm cov tshuaj tshwj xeeb. Li no, nws yog hu ua chemical sequencing. Sanger sequencing txoj kev tau qhia nyob rau hauv 1977, thiab yog ua raws li ddNTP uas tau tsav cov saw hlau txiav tawm. Sanger sequencing method nrov tshaj li Maxam Gilbert txoj kev vim muaj ntau yam tsis zoo ntawm Maxam Gilbert txoj kev xws li kev siv sijhawm ntau, siv tshuaj lom neeg, thiab lwm yam. Qhov no yog qhov txawv ntawm Maxam Gilbert thiab Sanger sequencing.
