Qhov Tseem Ceeb Qhov txawv - Forward vs Reverse Primer
Polymerase saw cov tshuaj tiv thaiv (PCR) yog DNA amplification txoj kev uas yog siv nyob rau hauv Molecular Biological daim ntaub ntawv. Nws yog ib qho txheej txheem uas feem ntau siv uas ua rau ntau lab mus rau billions ntawm cov ntawv luam ntawm cov DNA tshwj xeeb uas xav tau. Nws yog ib txoj hauv kev hauv vitro ua nyob rau hauv lub chaw soj nstuam. Cov txheej txheem PCR yog tag nrho nyob ntawm kev lag luam tsim DNA polymerase hu ua Taq polymerase. Thiab tseem nws yuav tsum muaj ob peb lwm yam khoom thiab kev saib xyuas kom zoo. Ib qho tseem ceeb tivthaiv yog primers. Primers yog cov luv luv DNA sequences tshwj xeeb uas tsim los rau lub hom phiaj DNA sib lawv liag. Lawv feem ntau nyob ib ncig ntawm 20 nucleotides ntev. Taq polymerase catalyzes ntxiv ntawm nucleotides rau hauv preexisting nucleotide sequence. Yog li ntawd, primers tau txais kev pab raws li pib cov ntsiab lus ntawm synthesis ntawm tshiab strands. Taq polymerase ua haujlwm tsuas yog nyob rau hauv 5' mus rau 3' kev taw qhia li no DNA synthesis tshwm sim nyob rau hauv tib 5' mus rau 3' kev taw qhia. Txij li DNA yog ob zaug tso tseg, ob hom primers xav tau hauv PCR. Lawv hu ua forward primer thiab thim rov qab primer. Forward thiab thim rov qab primers yog hu ua raws li cov kev taw qhia ntawm elongation ntawm primer hauv DNA thaum DNA synthesis tshwm sim. Forward primer anneals nrog antisense DNA strand thiab pib cov synthesis ntawm + ve strand ntawm cov noob rau hauv 5' rau 3' kev taw qhia. Rov qab primer anneals nrog lub siab strand thiab pib lub synthesis ntawm complementary strand ntawm lub coding strand; uas yog -ve lub strand ntawm cov noob rau hauv 5' rau 3' kev taw qhia. Qhov no yog qhov sib txawv tseem ceeb ntawm pem hauv ntej thiab rov qab primers.
Tus Thawj Fwm Tsav Tebchaws yog dab tsi?
Forward orientation yog kev sib txuas ntawm coding strand los yog qhov kev nkag siab ntawm cov noob. Taq polymerase catalyzes cov synthesis ntawm ib tug tshiab strand nyob rau hauv 5' rau 3' kev taw qhia. Lub synthesis ntawm coding strand tshwm sim thaum lub primer anneals nrog cov noncoding los yog antisense strand thiab elongates nyob rau hauv 5 'mus rau 3' kev taw qhia.
Daim duab 01: Forward and Reverse Primers
Tus primer uas anneals nrog antisense strand los yog noncoding strand los yog template strand yog lub npe hu ua forward primer txij li thaum pem hauv ntej primer ua raws li ib tug pib taw tes rau cov synthesis ntawm coding los yog lub zoo strand ntawm cov noob. Forward primer muaj ib tug luv luv nucleotide ib theem zuj zus uas yog ntxiv rau 3 'flnking kawg ntawm antisense strand. Nws hybridizes nrog antisense strand thiab pab txhawb lub Taq polymerase ntxiv nucleotides uas yog complementary rau lub template strand.
Dab tsi yog qhov rov qab primer?
Rov qab primer yog qhov luv luv DNA ntu uas anneals nrog 3' kawg ntawm qhov kev nkag siab strand lossis coding strand. Rov qab primer ua qhov pib taw tes rau kev sib txuas ua ke ntawm cov txheej txheem coding lossis qhov tsis sib xws. Rov qab primer yog tsim los ntxiv rau 3 'kawg ntawm coding strand. Li no, nws anneals nrog lub flanking 3 'kawg ntawm coding strand thiab tso cai rau Taq polymerase synthesize lub antisense strand los yog template strand. Txij li thaum nws txoj kev taw qhia yog nyob rau hauv ib qho kev thim rov qab, cov primer no tau sau tias yog thim rov qab primer.
Ob qho tib si thim rov qab thiab rau pem hauv ntej yog qhov tseem ceeb rau kev tsim ntau lab mus rau ntau lab luam ntawm cov cheeb tsam tshwj xeeb ntawm DNA uas tau tsom lossis nyiam.
Dab tsi zoo sib xws ntawm Forward thiab thim rov qab primer?
- Ob qho tib si Forward thiab thim rov qab primers yog tsim los ntawm oligonucleotides.
- Ob qho tib si rau pem hauv ntej thiab rov qab Primers muaj luv luv nucleotide ib ntus ntxiv rau qhov xaus ntawm DNA ob txoj hlua.
- Ob qho tib si Forward thiab thim rov qab primers feem ntau muaj 20 nucleotides.
- Ob qho tib si Forward thiab thim rov qab primers yog siv rau hauv cov tshuaj polymerase saw.
- Ob qho tib si Forward thiab thim rov qab Primers yog tsim los ua lag luam.
- Ob qho tib si Forward thiab thim rov qab primers yog qhov kub thiab txias thiab lawv ib txwm muaj qhov zoo sib xws Tm.
- Ob qho tib si Forward thiab thim rov qab Primers yog annealed nrog lub hom phiaj DNA sequences.
- Ob qho tib si rau pem hauv ntej thiab thim rov qab yog tsim los raws li PCR cov tshuaj tiv thaiv.
- Ob qho tib si Forward thiab thim rov qab Primers tau txais kev pabcuam raws li cov ntsiab lus pib rau DNA amplification.
- Ob qho tib si thim rov qab thiab rau pem hauv ntej yog qhov tseem ceeb rau kev tsim ntau lab luam ntawm cov cheeb tsam tshwj xeeb ntawm cov phiaj xwm lossis nyiam DNA ua ntu zus.
Qhov txawv ntawm Forward thiab thim rov qab primer yog dab tsi?
Forward Primer vs Reverse Primer |
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| Forward primer yog cov DNA luv luv uas sib txuas nrog 3 'kawg ntawm noncoding lossis tus qauv strand ntawm cov noob thiab ua haujlwm raws li qhov pib taw tes rau kev sib txuas ntawm coding sequence. | Rov qab primer yog qhov luv luv DNA ntu uas sib txuas nrog 3 'kawg ntawm cov coding lossis cov nontemplate strand thiab ua qhov pib taw tes rau kev sib txuas ntawm qhov tsis sib xws. |
| Annealing Strand | |
| Forward primer anneals nrog template strand. | Rov qab primer anneals nrog cov nontemplate strand. |
| Resulting New Sequence | |
| Forward primer pab txhawb kev sib txuas ntawm coding sequence. | Rov qab primer pab txhawb kev sib koom ua ke ntawm noncoding sequence. |
Summary – Forward vs Reverse Primer
Muaj ob hom primers koom nrog hauv cov txheej txheem PCR. Lawv yog cov pem hauv ntej thiab rov qab primers. Raws li qhov elongation ntawm primer nyob rau hauv tshiab DNA strand synthesis, cov primers yog sau los yog npe. Taq polymerase synthesizes DNA tshiab hauv 5' rau 3' orientation. Yog li ntawd, primers yog tsim raws li complementary rau 3 'kawg ntawm ob strands. Forward primer uas elongates nyob rau hauv 5 'mus rau 3' kev taw qhia hybridizes nrog 3 'kawg ntawm antisense los yog tus qauv los yog qhov tsis sib xws. Nws ua hauj lwm raws li qhov pib taw tes rau synthesize coding sequence. Rov qab primer uas elongates nyob rau hauv 5 'mus rau 3' kev taw qhia hybridizes nrog rau 3' kawg ntawm cov coding los yog lub nontemplate los yog lub siab strand. Nws ua hauj lwm raws li qhov pib taw tes rau synthesize noncoding sequence. Qhov no yog qhov sib txawv ntawm pem hauv ntej thiab rov qab primer.
