Mukuedza kweqPCR, dhizaini yekutanga zvakare yakakosha link.Ingave maprimers akakodzera kana kuti kwete ane hukama nepedyo nekuti iyo amplification inosvika pachiyero, ingave zvigadzirwa zvakasimudzirwa zvakanangana, uye kuti mhedzisiro yekuedza iripo.
Saka maitiro ekuita qPCR primer chaiyo zviri nani?High amplification kunyatsoshanda?
Nhasi, tichakutora iwe kugadzira qPCR maprimers pamwechete, uye rega qPCR primer dhizaini ive hunyanzvi hwekuita hunyanzvi mukuyedza.
Paunenge uchigadzira qPCR primers, kazhinji teerera kune zvinotevera pfungwa: maprimers anofanirwa kugadzirwa mhiri introns zvakanyanya sezvinobvira, kureba kwechigadzirwa kunofanira kunge kuri 100-300 bp, kukosha kweTm kunofanirwa kunge kuri padyo ne60 ° C, uye kumusoro uye kuzasi kwekutanga kunofanirwa kunge kuri padyo sezvinobvira, uye kuguma kwekutanga kunofanira kunge kuri G kana C, nezvimwewo kumirira.
1. Dhizaini yemaprimers inotenderera introns
Paunenge uchigadzira qPCR maprimers, kusarudza maprimers akagadzirwa mhiri maintrons anogona kudzivirira gDNA template kubva pakukwidziridzwa, uye zvigadzirwa zvese zvakatorwa kubva mukukwidziridzwa kwecDNA, nekudaro kubvisa pesvedzero yekusvibiswa kwegDNA.
2. Primer urefu
Iyo primer kureba kazhinji iri pakati pe18-30 nt, uye kureba kwechigadzirwa chekusimudzira kunofanirwa kudzorwa pakati pe100-300 bp zvakanyanya sezvinobvira.
Kana iyo primer yakanyanya kupfupika, inotungamira kune isiri-yakananga amplification, uye kana yakanyanya kureba, ichaita nyore nyore chimiro chechipiri (senge hairpin chimiro).Kana chigadzirwa chekusimudzira chakareba, hachina kukodzera kuita kwepolymerase, izvo zvinokanganisa kushanda kwePCR kukwidziridzwa.
3. GC zvemukati uye Tm kukosha
Iyo GC yemukati yemaprimers inofanirwa kudzorwa pakati pe40% ne60%.Kana yakanyanyisa kana kuti yakaderera, hazvibatsiri kutanga kuita.Iyo GC yemukati meyekumberi uye yekudzosera maprimers inofanirwa kunge iri padyo neyakafanana kuti iwane iyo yakafanana Tm kukosha uye annealing tembiricha.
Huwandu hweTm hunofanirwa kunge huri pakati pe55-65°C kusvika pazvinobvira, kazhinji kutenderera 60°C, uye kukosha kweTm kwekumusoro nekudzika kunofanira kunge kuri padyo sezvinobvira, zvichida kusapfuure 4°C.
4. Dzivisa kusarudza A pa3 ′ kumagumo ekutanga
Kana iyo 3 ′ kuguma kweprimer isingaenderane, kune misiyano mikuru mukuita kwe synthesis yemabhesi akasiyana.Apo chigadziko chekupedzisira chiri A, chinogonawo kutanga chain synthesis kunyange munyaya yekusawirirana, uye kana chigadziko chekupedzisira chiri T Kana , kushanda kwekusawirirana induction kunoderedzwa zvikuru.Naizvozvo, edza kudzivirira kusarudza A pa3 ′ kumagumo ekutanga, uye zviri nani kusarudza T.
Kana iri probe primer, iyo 5' kuguma kweprobe haigoni kuva G, nokuti kunyange kana imwe G base yakabatana neboka revatori venhau reFAM fluorescent, G inogonawo kudzima chiratidzo chefluorescent chakabudiswa neboka reFAM, zvichiita kuti pave nemigumisiro yakaipa yenhema.Kuonekwa.
5. Base distribution
Kugoverwa kwezvigadziko zvina muprimer zviri nani zvisingaite, kudzivirira zvinopfuura 3 zvakatevedzana G kana C kumagumo e3, uye anopfuura matatu akateedzana.G kana C zviri nyore kugadzira pairing munharaunda yeGC-hupfumi hwekutevedzana.
6. Iyo primer dhizaini dunhu inofanirwa kudzivirira yakaoma yechipiri zvimiro.
Iyo yechipiri chimiro chakaumbwa neicho tambo yechigadzirwa chekusimudzira chinozokanganisa kufambira mberi kwakanaka kwePCR.Nekufanotaura kana paine chechipiri chimiro mukutevedzana kwakanangana mberi, edza kudzivirira dunhu iri mukugadzirwa kwekutanga.
7. Iwo maprimer pachawo uye pakati pemaprimers anofanirwa kuyedza kudzivirira anotevedzana mabhesi ekuwedzera.
Ikoko hakugone kuve nekuteedzana 4 base kuwirirana pakati peiyo primer pachayo uye yekutanga.Iyo primer pachayo haifanirwe kuve neanopindirana kutevedzana, zvikasadaro inozvipeta pachayo kuti iite hairpin chimiro, icho chinozokanganisa kusanganisa kweannealing yeprimer uye template.
Kutevedzana kunotevedzana hakugone kuvapo pakati pemaprimers ekukwira nekudzika.Kupindirana pakati pemaprimers kunoburitsa primer dimers, iyo inoderedza PCR kunyatsoita uye kunyange kukanganisa kuwanda kwehuwandu.Kana iyo primer-dimer uye hairpin zvimiro zvisingadzivisike, iyo △G kukosha haifanirwe kunge yakanyanya kukwirira (inofanira kunge iri pasi pe4.5 kcal/mol).
8. Iwo maprimers anowedzera chinangwa chaicho chigadzirwa.
Chinangwa chekupedzisira chekuonekwa kweqPCR ndechekunzwisisa kuwanda kweiyo gene gene.Kana isiri-chaiyo amplification ikaitika, iyo quantification ichave isina kururama.Naizvozvo, mushure mekunge maprimers agadzirwa, anofanirwa kuyedzwa neBLAST, uye iwo chaiwo ezvigadzirwa anofananidzwa mune inoteedzana dhatabhesi.
Tevere, tinotora iyo GAS6 yemunhu (Kukura kusungwa chaiko 6) gene semuenzaniso wekugadzira qPCR primers.
01 mubvunzo gene
Homo GAS6kuburikidza neNCBI.Pano, tinofanira kutarisisa kuenzanisa zita rejena uye mhando kuti tive nechokwadi chekuti zvinoenderana.
02 Tsvaga kutevedzana kwemajini
(1) Kana chinangwa chakatevedzana chiri genomic DNA, sarudza yekutanga, inova genomic DNA sequence yejini.
(2) Kana chinangwa chekutevedzana chiri mRNA, sarudza yechipiri.Mushure mekupinda, tinya "CDS" mutafura iri pazasi.Kutevedzana kwebrown kumashure ndiko kutevedzana kwekodha yejini.
03 Dhizaini yekutanga
Pinda iyo Primer-BLAST interface
Isa iyo gene sequence nhamba kana iyo inoteedzana muFasta fomati kumusoro kuruboshwe, uye zadza akakodzera paramita.

Dzvanya "Tora maprimers" uye NCBI ichabuda kuti ikuudze kuti kusarudzwa kwakadaro kweparameter kuchakwidziridzwa kune mamwe ma spicing akasiyana.Tinogona kutarisa akasiyana splicing akasiyana uye toaendesa kuti titore yakakodzera primer pair (sezvinoratidzwa pamufananidzo uri pazasi).Iyi nzira inogona kutora makumi emasekonzi kuti iite.
Tembiricha dzemaprimer pairs idzi dzakatenderedza 60°C.Zvinoenderana nechinangwa chekuyedza, sarudza maprimers ane hurefu huri pakati nepakati, yakanaka chaiyo uye kushoma kuzvizadzisa kwekutanga kwekuyedza, uye mwero wekubudirira wakanyanya kukwirira!
04Primer chaiyo verification
Muchokwadi, pamusoro pekugadzira maprimers, Primer-Blast inogona zvakare kuongorora maprimers atakagadzira isu pachedu.Dzokera kune yekutanga dhizaini peji, pinda kumusoro uye pasi pemvura maprimers atakagadzira, uye mamwe ma paramita haagadziriswe.Mushure mekutumira, unogona kuona kana maviri ekutanga aripowo pane mamwe majini.Kana ese akaratidzwa pane jena ratinoda kukwidziridza, zvichiratidza kuti iyo chaiyo yeiyi mbiri yekutanga yakanaka!(Semuenzaniso, iyi ndiyo yega mhedzisiro yemubvunzo wekutanga!)

05 Kutonga kwemhando yepamusoro
Ndeupi rudzi rwekutanga iyo "yakakwana" primer inosanganisa "amplification kunyatsoita kusvika kune yakajairwa", "amplified chigadzirwa maitiro", uye "yakavimbika mhedzisiro yekuyedza"?
Amplification kunyatsoshanda

melting curve
Kubudirira kwekusimudzira kwemaprimers kunosvika 90% -110%, izvo zvinoreva kuti kukwidziridzwa kwakanaka kwakanaka, uye kunyunguduka kunonyunguduka kune imwe peak uye kazhinji Tm> 80 ° C, zvinoreva kuti chirevo chekusimudzira chakanaka.
 
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