Qhov Tseem Ceeb Qhov txawv - PCR Primers vs Sequencing Primers
Nrog cov kev txhim kho tsis ntev los no hauv thaj chaw ntawm molecular biology, cov txheej txheem caj ces sib txawv tau tsim los ua kom cov txheej txheem tshawb xyuas ntawm cov txheej txheem sib txawv ntawm cov ncauj lus tau yooj yim thiab raug. PCR thiab lwm cov txheej txheem sequencing yog ob qho tseem ceeb xws li cov txheej txheem. Lawv siv cov khoom sib txawv. Primers raug suav hais tias yog cov ntsiab lus tseem ceeb ntawm ob qho tib si PCR thiab Sequencing cov txheej txheem. PCR primers yog siv rau kev nthuav dav ntawm ib qho DNA ib ntus thaum lub sij hawm sequencing primers yog siv nyob rau hauv cov ntsiab lus ntawm sequencing DNA fragment nrog lub hom phiaj ntawm nthuav qhia nws cov kev txiav txim tshwj xeeb ntawm nucleotide sequence. Qhov no yog qhov sib txawv tseem ceeb ntawm PCR primers thiab sequencing primers.
PCR Primers yog dab tsi?
Polymerase Chain Reaction (PCR) yog cov txheej txheem caj ces uas tau siv nyob rau hauv thaj chaw ntawm molecular biology txhawm rau txhawm rau nthuav dav ib lossis ob peb daim ntawv luam ntawm ib ntu DNA tshwj xeeb thiab kom tau txais ntau lab ntawm cov ntawv luam zoo ib yam. Hauv PCR cov tshuaj tiv thaiv, cov khoom sib txawv yog siv nrog rau cov primers. Primers yog luv DNA strands nrog ib tug nucleotide ntev ntawm 18-25 ua rau lawv sib xws nrog rau qhov pib thiab qhov kawg cheeb tsam ntawm DNA fragments kom amplified. Primers tuaj yeem ua rau pem hauv ntej primer thiab rov qab primer. Cov primers khi rau DNA tawg ntawm cov ntsiab lus tshwj xeeb uas nws ua rau DNA polymerase los khi rau cov primer tshwj xeeb ntawm qhov chaw thiab pib ua cov synthesis ntawm DNA tshiab strand.
Kev xaiv cov primers yog ib qho tseem ceeb ntawm cov txheej txheem PCR. Kev xaiv qhov ntev ntawm primer yog qhov tseem ceeb. Qhov zoo tagnrho ntev yuav yog 18-25 nucleotides. Yog tias qhov ntev luv los yog ntev dhau lawm, cov primers yuav tsis khi rau DNA ib ntus kom tau txais kev nthuav dav kom raug. Primers uas luv luv ntev ua rau tsis muaj qhov tshwj xeeb primer annealing ntawm qhov chaw sib txawv ntawm DNA sib lawv liag.
Daim duab 01: PCR Primers
Cov ntsiab lus Guanine thiab Cytosine (GC) hauv cov primer zoo yuav tsum nyob rau hauv thaj tsam ntawm 40-60. Lub primer annealing kub thiab melting kub yog qhov tseem ceeb ntawm PCR. Lub melting kub yuav tsum raug xam kom raug, thiab cov primer annealing kub yuav tsum yog 5 0 C tsawg dua qhov melting kub. Lub melting kub yuav tsum yog 60 ° C thiab 75 ° C. Qhov kub thiab txias dhau lawm yuav ua rau muaj kev ua haujlwm ntawm DNA polymerase tsawg dua.
Dab tsi yog Sequencing Primers?
Sequencing primers yog siv nyob rau hauv cov ntsiab lus ntawm sequencing DNA fragment nrog rau lub hom phiaj ntawm nthuav qhia nws tus kheej tshwj xeeb. Txhawm rau kom tau txais txiaj ntsig zoo sib luag zoo siab primers thiab cov qauv yog qhov tseem ceeb. Yog li, thaum xaiv primers, lawv yuav tsum yog tshwj xeeb rau ib cheeb tsam uas peb xav kom ua raws. Nws kuj yuav tsum yog nrog kev taw qhia kom raug qhov twg cov kab ke feem ntau yog tsim los ntawm 3' mus rau 5' kawg ntawm cov primers. Qhov sib lawv liag yuav tsum tsis muaj undesirable self-hybridization xws li tsim ntawm hairpin loops. Nws yuav tsum tsis txhob muaj qhov sib law liag ntawm Guanine bases.
Lub melting kub (Tm) ntawm primer yuav tsum haum rau cov xwm txheej ntawm sequencing. Yog li ntawd, nws yuav tsum dag ntawm 52oC thiab 74oC. Kev npaj ntawm oligonucleotides siv los ua primer yuav tsum tau ua kom huv si kom tau txais qhov xav tau tag nrho ntawm qhov sib lawv liag. Yog hais tias cov oligonucleotides muaj impurities, lub primer sequence signaling yuav superimposed los ntawm txawv priming sites, thiab nws kuj yuav txo tus naj npawb ntawm cov hauv paus hlwb.
Daim duab 02: Sequencing Primers
Lub primer melting kub (Tm) ntawm ib qho oligonucleotide txiav txim siab, muaj zog npaum li cas cov DNA txuas ntxiv yog sib xyaw nrog ib leeg. Tm tuaj yeem suav tau tias yog kev ntsuas ntsuas qhov ntsuas kub qhov twg nws nyob ntawm ob qho tib si DNA thiab ntau yam xwm txheej xws li ntsev concentration. Lub Tm yog ib qho tseem ceeb thaum lub sij hawm PCR qhov twg ib tug variant hu ua lub voj voog sequencing yog siv los tsim ib pab pawg neeg ntawm dideoxynucleotide-terminated fragments. Ntawm no, cov primer uas yog sequenced yuav pib yuav annealed hloov lwm lub tsev, ces ncua thiab zaum kawg denatured rau amplification. Yog li ntawd, tus nqi Tm yuav tsum nyob nruab nrab ntawm 52oC thiab 74o C. Synthesized oligonucleotides tuaj yeem tau txais los ntawm DNA / RNA synthesis laboratories raws li ib tug kev xaiv. Qhov me me ntawm kev sib txuas uas yog siv rau DNA sequencing feem ntau yog 50 nmol. Tsis tas li ntawd tseem ceeb tshaj plaws cov primers siv rau sequencing yuav tsum tau purified kom tsis muaj impurities uas yuav tiv thaiv tau qhov zoo txo.
Dab tsi yog qhov zoo sib xws ntawm PCR Primers thiab Sequencing Primers?
- Ob qho tib si PCR Primers thiab Sequencing Primers yog cov primers uas tau siv rau hauv cov txheej txheem nthuav dav ntawm cov phiaj xwm DNA.
- Ob lub PCR Primers thiab Sequencing Primers yog tsim los ntawm nucleotides.
- Ob leeg PCR Primers thiab Sequencing Primers yog luv oligomers.
Qhov txawv ntawm PCR Primers thiab Sequencing Primers yog dab tsi?
PCR Primers vs Sequencing Primers |
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| PCR primers yog luv DNA strands nrog cov nucleotide ib ntu ntev ntawm 18-25 ua rau lawv sib haum nrog qhov pib thiab qhov kawg cheeb tsam ntawm DNA tawg uas yuav tsum tau nthuav dav. | Sequencing primers yog luv oligomers uas yog siv nyob rau hauv lub ntsiab lus ntawm sequencing ib DNA tawg nrog lub hom phiaj ntawm kev nthuav tawm nws tus kheej tshwj xeeb. |
| Muaj nuj nqi | |
| PCR primers yog siv rau kev nthuav dav ntawm DNA ib ntus. | Sequencing primers yog siv nyob rau hauv cov ntsiab lus ntawm sequencing DNA fragment nrog rau lub hom phiaj ntawm nthuav qhia nws tus kheej tshwj xeeb. |
| Ntau tus primers Xav tau | |
| Ob tug primers; ib qho primer rau pem hauv ntej thiab ib qho rov qab primer yog siv los ua PCR primers. | Xav tau ib qho primer li sequencing primer. |
Summary – PCR Primers vs Sequencing Primers
Sequencing primers yog siv nyob rau hauv cov ntsiab lus ntawm sequencing DNA fragment nrog rau lub hom phiaj ntawm nthuav qhia nws tus kheej tshwj xeeb. Ib tug sequencing primer yuav txaus los khiav cov txheej txheem. Txhawm rau kom tau txais txiaj ntsig zoo, cov primers zoo thiab cov qauv yog qhov tseem ceeb. Yog li, thaum xaiv primers, lawv yuav tsum yog tshwj xeeb rau ib cheeb tsam uas peb xav kom ua raws. PCR Primers yog luv DNA strands nrog ib tug nucleotide ntev ntawm 18-25 uas yog sib xws nrog rau qhov pib thiab qhov kawg cheeb tsam ntawm DNA fragments uas yuav tsum tau amplified. PCR primers tuaj yeem ua rau pem hauv ntej primer thiab thim rov qab primer. Cov ntsiab lus ntawm Guanine thiab Cytosine (GC) nyob rau hauv ib qho zoo primer yuav tsum nyob rau hauv thaj tsam ntawm 40-60. Lub primer annealing kub thiab melting kub yog qhov tseem ceeb thaum lub sij hawm PCR. Qhov no yog qhov txawv ntawm PCR primers thiab Sequencing primers.
