Mubigeragezo bya qPCR, primer igishushanyo nacyo gihuza cyane.Niba primers ikwiye cyangwa idakwiye bifitanye isano ya hafi niba imikorere ya amplification igera ku gipimo, niba ibicuruzwa byongerewe umwihariko, kandi niba ibisubizo byubushakashatsi bihari.
Nigute ushobora gukora qPCR primer yihariye?Gukora neza cyane?
Uyu munsi, tuzagutwara gushushanya qPCR primers hamwe, hanyuma tureke igishushanyo cya qPCR gihinduke ubuhanga bwa lore mubushakashatsi.
Mugihe utegura primaire ya qPCR, mubisanzwe witondere ingingo zikurikira: primers igomba kuba yarateguwe murwego rwinshi rushoboka, uburebure bwibicuruzwa bugomba kuba 100-300 bp, agaciro ka Tm kagomba kuba hafi gashoboka kuri 60 ° C, naho primers yo hejuru no kumanuka igomba kuba hafi bishoboka, kandi iherezo rya primer rigomba kuba G cyangwa C, nibindi gutegereza.
1. Igishushanyo cya primers kizenguruka intangiriro
Mugushushanya primers ya qPCR, guhitamo primers zakozwe murwego rwimbere birashobora kubuza inyandikorugero ya gDNA kwongerwaho, kandi ibicuruzwa byose biva mugukwirakwiza cDNA, bityo bikuraho ingaruka ziterwa na gDNA.
2. Uburebure bwa primer
Uburebure bwa primer muri rusange buri hagati ya 18-30 nt, kandi uburebure bwibicuruzwa byongerewe imbaraga bugomba kugenzurwa hagati ya 100-300 bp bishoboka.
Niba primer ari ngufi cyane, bizaganisha kuri amplification idasanzwe, kandi niba ari ndende cyane, izahita ikora urwego rwa kabiri (nkimiterere yimisatsi).Niba ibicuruzwa byongerewe imbaraga ari birebire, ntibikwiye kubyitwaramo polymerase, bizagira ingaruka kumikorere ya PCR.
3. Ibirimo GC nagaciro ka Tm
GC yibigize primers igomba kugenzurwa hagati ya 40% na 60%.Niba ari hejuru cyane cyangwa hasi cyane, ntabwo bifasha gutangiza reaction.Ibiri muri GC byimbere nibisubira inyuma bigomba kuba hafi kimwe kugirango tubone agaciro ka Tm hamwe nubushyuhe bwa annealing.
Agaciro ka Tm kagomba kuba hagati ya 55-65 ° C uko bishoboka kose, muri rusange hafi 60 ° C, kandi agaciro ka Tm kumugezi wo hejuru no kumanuka bigomba kuba hafi bishoboka, byaba byiza bitarenze 4 ° C.
4. Irinde guhitamo A kuri 3 ′ iherezo rya primer
Iyo 3 ′ iherezo rya primer idahuye, hari itandukaniro rikomeye muburyo bwo guhuza imikorere yibikorwa bitandukanye.Iyo ishingiro ryanyuma ari A, rirashobora kandi gutangiza urunigi rwumunyururu nubwo haba rudahuye, kandi mugihe shingiro ryanyuma ari T Iyo, imikorere yo kwinjiza idahuye iragabanuka cyane.Noneho, gerageza wirinde guhitamo A kuri 3 ′ iherezo rya primer, kandi nibyiza guhitamo T.
Niba ari primer primer, 5 ′ iherezo ryiperereza ntirishobora kuba G, kuko niyo mugihe G base imwe ihujwe nitsinda ryabanyamakuru ba FAM fluorescent, G irashobora kandi kuzimya ibimenyetso bya fluorescente byatanzwe nitsinda rya FAM, bikavamo ibisubizo bibi.Kugaragara.
5. Gukwirakwiza shingiro
Ikwirakwizwa ryibice bine muri primer nibyiza guhitamo, wirinda ibirenga 3 bikurikiranye G cyangwa C kuri 3 ′, kandi birenze 3 bikurikiranye.G cyangwa C biroroshye kubyara byombi mukarere gakungahaye kuri GC.
6. Igishushanyo mbonera cya primer kigomba kwirinda ibyiciro byisumbuyeho.
Imiterere ya kabiri yashizweho numurongo umwe wibicuruzwa byongera imbaraga bizagira ingaruka kumajyambere ya PCR.Muguhanura niba hari urwego rwa kabiri murwego rwateganijwe mbere, gerageza wirinde kariya karere mugushushanya primers.
7. Intangiriro ubwayo no hagati ya primers igomba kugerageza kwirinda ishingiro ryuzuzanya.
Ntabwo hashobora kubaho ibyingenzi 4 byuzuzanya hagati ya primer ubwayo na primer.Primer ubwayo ntigomba kugira urutonde rwuzuzanya, bitabaye ibyo izikubye kugirango ikore imisatsi yimisatsi, izagira ingaruka kuri annealing ihuza primer hamwe nicyitegererezo.
Urutonde rwuzuzanya ntirushobora kubaho hagati yimbere na epfo na ruguru.Kwuzuzanya hagati ya primers bizatanga primer dimers, bizagabanya imikorere ya PCR ndetse bigire ingaruka kumubare wuzuye.Niba primer-dimer na hairpin ibyubaka bidashobora kwirindwa, value G agaciro ntigomba kuba hejuru cyane (igomba kuba munsi ya 4.5 kcal / mol).
8. Intangiriro yongerera intego ibicuruzwa byihariye.
Intego nyamukuru yo kumenya qPCR ni ukumva ubwinshi bwa gen igenewe.Niba amplification idasanzwe ibaye, ingano izaba idahwitse.Kubwibyo, nyuma ya primers zateguwe, zigomba kugeragezwa na BLAST, kandi umwihariko wibicuruzwa ugereranijwe mububiko bukurikirana.
Ibikurikira, dufata gene GAS6 yumuntu (Gukura gukura byihariye 6) nkurugero rwo gushushanya qPCR primers.
01 ikibazo
Homo GAS6binyuze muri NCBI.Hano, dukwiye kwitondera kugereranya izina rya gen nubwoko kugirango tumenye ko bihuye.
02 Shakisha gene ikurikirana
(1) Niba intego ikurikiranye ari ADN genomic, hitamo iyambere, aribwo ADN ikurikirana ya gen.
(2) Niba intego ikurikiranye ari mRNA, hitamo iyakabiri.Nyuma yo kwinjira, kanda "CDS" mumeza hepfo.Urukurikirane rw'ibara ry'umukara ni code ya gen ikurikirana.
03 Shushanya primers
Injira Primer-BLAST Imigaragarire
Injiza gene ikurikirana cyangwa ikurikiranye muburyo bwa Fasta ibumoso bwo hejuru, hanyuma wuzuze ibipimo bijyanye.

Kanda "Get primers" hanyuma NCBI izaduka kugirango ikubwire ko guhitamo ibipimo bizongerwaho kubindi bitandukanya.Turashobora kugenzura ibintu bitandukanye bitandukanye hanyuma tukabitanga kugirango tubone primer couple ikwiye (nkuko bigaragara mumashusho hepfo).Iyi nzira irashobora gufata amasegonda icumi kugirango ikore.
Ubushyuhe bwa annealing bwaba primer ni hafi 60 ° C.Ukurikije intego yubushakashatsi, hitamo primers ifite uburebure buringaniye, umwihariko mwiza no kutuzuzanya kwa primers kubigeragezo, kandi intsinzi iri hejuru cyane!
04Igenzura ryihariye
Mubyukuri, usibye gushushanya primers, Primer-Blast irashobora kandi gusuzuma primers twashizeho ubwacu.Garuka kurupapuro rwibishushanyo mbonera, andika epfo na ruguru primers twashizeho, nibindi bipimo ntabwo bizahinduka.Nyuma yo gutanga, urashobora kureba niba couple ya primers nayo ibaho kurindi gen.Niba byose byerekanwe kuri gene dushaka kongera, byerekana ko umwihariko wiyi couple ya primers ari nziza!(Kurugero, iki nigisubizo cyonyine cyibibazo bya primer!)

05 Urubanza rwiza
Ni ubuhe bwoko bwa primer "primer" primer ihuza "amplification efficient to standard", "ibicuruzwa byongerewe ibicuruzwa", n "ibisubizo byizewe byubushakashatsi"?
Gukora neza

gushonga umurongo
Gukora neza kwa primers bigera kuri 90% -110%, bivuze ko imikorere ya amplification ari nziza, kandi umurongo wo gushonga ufite impinga imwe kandi mubisanzwe Tm> 80 ° C, bivuze ko umwihariko wa amplification ari mwiza.
 
Ibicuruzwa bifitanye isano:
Igihe nyacyo PCR Byoroshye - SYBR GREEN I.
Igihe nyacyo PCR Byoroshye-Taqman