Qhov Tseem Ceeb Qhov txawv - Sanger Sequencing vs Pyrosequencing
DNA sequencing yog ib qho tseem ceeb heev rau kev soj ntsuam DNA vim kev paub txog qhov tseeb nucleotides kev npaj ntawm ib cheeb tsam DNA qhia ntau cov ntaub ntawv tseem ceeb txog nws. Muaj cov txheej txheem DNA sib txawv. Sanger sequencing thiab Pyrosequencing yog ob txoj kev sib txawv DNA sib txawv siv dav hauv Molecular Biology. Qhov sib txawv tseem ceeb ntawm Sanger sequencing thiab Pyrosequencing yog tias Sanger sequencing siv dideoxynucleotides los txiav tawm cov synthesis ntawm DNA los nyeem cov nucleotide sequence thaum pyrosequencing pom cov pyrophosphate tso tawm los ntawm kev sib koom ua ke ntawm nucleotides thiab kev sib txuas ua ke ntawm kev nyeem ntawv ua ntej.
Sanger Sequencing yog dab tsi?
Sanger sequencing yog thawj tiam DNA sequencing txoj kev tsim los ntawm Frederick Sanger thiab nws cov tsev kawm ntawv qib siab hauv xyoo 1977. Nws tseem hu ua Chain Termination Sequencing lossis Dideoxy sequencing vim nws yog raws li cov saw txiav los ntawm Dideoxynucleotides (ddNTPs). Txoj kev no tau siv dav rau ntau tshaj 30 xyoo kom txog rau thaum New Generation Sequencing (NGS) tau tsim. Sanger sequencing txheej txheem tau qhib qhov kev tshawb pom ntawm qhov tseeb nucleotide kev txiav txim lossis txuas ntawm ib qho DNA feem. Nws yog raws li kev xaiv koom ua ke ntawm ddNTPs thiab kev txiav tawm ntawm DNA synthesis thaum lub sij hawm in vitro DNA replication. Qhov tsis muaj 3 'OH pawg los txuas ntxiv phosphodiester daim ntawv cog lus ntawm cov nucleotides uas nyob ib sab yog ib qho tshwj xeeb ntawm ddNTPs. Li no, thaum lub ddNTP txuas nrog, saw elongation ceases thiab terminates los ntawm qhov ntawd. Muaj plaub ddNTPs - ddATP, ddCTP, ddGTP thiab ddTTP - siv hauv Sanger sequencing. Cov nucleotides nres cov txheej txheem DNA rov ua dua thaum lawv tau muab tso rau hauv qhov loj hlob ntawm DNA thiab ua rau muaj qhov sib txawv ntawm qhov ntev ntawm DNA. Capillary gel electrophoresis yog siv los npaj cov DNA luv luv los ntawm lawv qhov ntau thiab tsawg ntawm cov gel raws li qhia hauv daim duab 01.
Daim duab 1: Capillary gel electrophoresis ntawm synthesized luv DNA
Rau qhov kev hloov pauv hauv vitro ntawm DNA, yuav tsum tau muab ob peb yam. Lawv yog DNA polymerase enzyme, qauv DNA, oligonucleotide primers, thiab deoxynucleotides (dNTPs). Hauv Sanger sequencing, DNA replication yog ua nyob rau hauv plaub cais xeem tub nrog rau plaub hom ddNTPs nyias. Deoxynucleotides tsis tas hloov los ntawm cov ddNTPs. Ib qho kev sib xyaw ntawm dNTP tshwj xeeb (piv txwv li; dATP + ddATP) suav nrog hauv lub raj thiab rov ua dua. Plaub cov khoom sib cais yog khiav ntawm ib lub gel hauv plaub qhov dej cais. Tom qab ntawd los ntawm kev nyeem cov gel, cov kab ke tuaj yeem tsim tau raws li qhia hauv daim duab 02.
Daim duab 02: Sanger sequencing
Sanger sequencing yog cov txheej txheem tseem ceeb uas pab hauv ntau qhov chaw ntawm molecular biology. Tib neeg txoj haujlwm genome tau ua tiav tiav nrog kev pab ntawm Sanger sequencing-based txoj kev. Sanger sequencing kuj tseem siv tau rau hauv hom DNA sequencing, kev tshawb fawb mob qog noj ntshav thiab kab mob caj ces, kev tshuaj ntsuam gene, kev txheeb xyuas tib neeg, kuaj kab mob, kab mob microbial sequencing thiab lwm yam.
Muaj ntau qhov tsis zoo ntawm Sanger sequencing:
- Qhov ntev ntawm DNA tau ua ntu zus tsis tuaj yeem ntev dua 1000 lub hauv paus khub
- Tsuas yog ib txoj hlua tuaj yeem ua raws ib zaug.
- txheej txheem siv sijhawm thiab kim.
Yog li ntawd, cov txheej txheem tshiab ua ntu zus tau tsim nrog lub sijhawm los daws cov teeb meem no. Txawm li cas los xij, Sanger sequencing tseem siv tau vim nws cov txiaj ntsig tau raug siab txog li 850 lub hauv paus khub ntev fragments.
Pyrosequencing yog dab tsi?
Pyrosequencing yog cov txheej txheem DNA tshiab ua raws li "kev sib txuas los ntawm kev sib txuas". Cov txheej txheem no tso siab rau kev tshawb pom ntawm pyrophosphate tso tawm raws li kev koom ua ke ntawm nucleotide. Cov txheej txheem yog ua haujlwm los ntawm plaub yam enzymes: DNA polymerse, ATP sulfurylase, luciferase thiab apyrase thiab ob lub substrates adenosine 5' phosphosulfate (APS) thiab luciferin.
Cov txheej txheem pib nrog cov primer khi nrog ib leeg-stranded DNA qauv thiab DNA polymerase pib kev koom ua ke ntawm nucleotides ntxiv rau nws. Thaum cov nucleotides koom ua ke (nucleic acid polymerization), nws tso tawm pyrophosphate (ob pawg phosphate ua ke) pawg thiab lub zog. Txhua nucleotide ntxiv tso tawm qhov sib npaug ntawm pyrophosphate. Pyrophosphate hloov mus rau hauv ATP los ntawm ATP sulfurylase nyob rau hauv lub xub ntiag ntawm substrate APS. Lub generated ATP tsav lub luciferase-mediated hloov dua siab tshiab ntawm luciferin rau oxyluciferin, ua pom lub teeb nyob rau hauv cov nqi uas yog proportional rau tus nqi ntawm ATPs. Lub teeb raug kuaj pom los ntawm lub tshuab tshawb pom photon lossis los ntawm photomultiplier thiab tsim cov pyrogram. Apyrase degrades ATP thiab non-incorporated dNTPs hauv cov tshuaj tiv thaiv sib tov. dNTP ntxiv yog ua ib zaug ib zaug. Txij li thaum qhov sib ntxiv ntawm nucleotide paub raws li kev koom ua ke thiab kev tshawb pom ntawm lub teeb, qhov sib lawv liag ntawm cov qauv tuaj yeem txiav txim siab. Pyrogram yog siv los tsim cov nucleotide ib ntus ntawm cov qauv DNA raws li qhia hauv daim duab 03.
Pyrosequencing yog ib qho tseem ceeb heev nyob rau hauv ib qho nucleotide polymorphism tsom xam thiab sequencing ntawm luv stretches ntawm DNA. Qhov raug siab, yoog raws, yooj yim ntawm automation thiab ua tib yam yog qhov zoo ntawm pyrosequencing tshaj Sanger sequencing cov tswv yim.
Daim duab 03: Pyrosequencing
Dab tsi yog qhov txawv ntawm Sanger Sequencing thiab Pyrosequencing?
Sanger Sequencing vs Pyrosequencing |
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| Sanger sequencing yog ib txoj hauv kev DNA sequencing raws li kev xaiv ntawm ddNTPs los ntawm DNA polymerase thiab saw termination. | Pyrosequencing yog ib txoj hauv kev DNA sequencing raws li kev tshawb pom ntawm pyrophosphate tso tawm raws li kev koom ua ke ntawm nucleotide. |
| Siv ddNTP | |
| ddNTPs yog siv los txiav tawm DNA replication | ddNTPs tsis siv. |
| Enzymes koom nrog | |
| DNA polymerase yog siv. | Plaub enzymes siv: DNA polymerase, ATP sulfurylase, Luciferase thiab Apyrase. |
| siv cov substrates | |
| APS thiab Luciferin tsis siv. | Adenosine 5' phosphosulfate (APS) thiab luciferin yog siv. |
| siab kub | |
| Nov yog txheej txheem qeeb. | Nov yog txheej txheem ceev. |
Summary – Sanger Sequencing vs Pyrosequencing
Sanger sequencing thiab Pyrosequencing yog ob txoj kev DNA sequencing siv hauv molecular biology. Sanger sequencing tsim qhov kev txiav txim ntawm cov nucleotides nyob rau hauv ib theem zuj zus los ntawm kev txiav cov saw elongation thaum lub pyrosequencing tsim lub meej kev txiav txim ntawm nucleotides nyob rau hauv ib ntus los ntawm kev koom ua ke ntawm nucleotides thiab kuaj pom qhov tso tawm ntawm pyrophosphates. Yog li ntawd, qhov sib txawv tseem ceeb ntawm Sanger sequencing thiab Pyrosequencing yog tias Sanger sequencing ua haujlwm ntawm kev sib cais los ntawm cov saw txiav thaum pyrosequencing ua haujlwm ntawm sequencing los ntawm synthesis.
