Pakuyesa kwa qPCR, mapangidwe oyambira nawonso ndi ulalo wofunikira kwambiri.Kaya zoyambira zili zoyenera kapena ayi zimagwirizana kwambiri ndi ngati kukulitsa bwino kumafika pamlingo, kaya zokulitsa zili zenizeni, komanso ngati zotsatira zoyeserera zilipo.
Ndiye mungapangire bwanji mawonekedwe a qPCR oyambira bwino?High amplification mphamvu?
Lero, tikutengerani kuti mupange zoyambira za qPCR palimodzi, ndikulola mapangidwe oyambira a qPCR kukhala luso lodziwika bwino pakuyesa.
Popanga zoyambira za qPCR, nthawi zambiri samalani ndi mfundo zotsatirazi: zoyambira ziyenera kupangidwa modutsa ma introns momwe zingathere, kutalika kwa mankhwala kuyenera kukhala 100-300 bp, mtengo wa Tm uyenera kukhala pafupi kwambiri ndi 60 ° C, ndipo zoyambira kumtunda ndi kumunsi ziyenera kukhala pafupi momwe zingathere, ndipo mapeto a choyambira ayenera kukhala G kapena C, ndi zina zotero.
1. Kupanga koyambira koyambira kumayambira
Popanga zoyambira za qPCR, kusankha zoyambira zomwe zidapangidwa kudutsa ma introns zitha kuletsa template ya gDNA kuti isakulitsidwe, ndipo zinthu zonse zimachokera ku kukulitsa kwa cDNA, motero kuthetsa kukhudzidwa kwa kuipitsidwa kwa gDNA.
2. Kutalika koyambira
Kutalika koyambira nthawi zambiri kumakhala pakati pa 18-30 nt, ndipo kutalika kwa chinthu chokulitsa kuyenera kuyendetsedwa pakati pa 100-300 bp momwe ndingathere.
Ngati choyambiracho ndi chachifupi kwambiri, chimayambitsa kukulitsa kopanda tanthauzo, ndipo ngati kuli kotalika kwambiri, kumapanga mawonekedwe achiwiri (monga mawonekedwe a hairpin).Ngati mankhwala okulitsa ndi otalika kwambiri, siwoyenera kuyankha kwa polymerase, zomwe zingakhudze mphamvu ya kukulitsa kwa PCR.
3. GC zili ndi mtengo wa Tm
Zomwe zili mu GC zoyambira ziyenera kuyendetsedwa pakati pa 40% ndi 60%.Ngati ndi okwera kwambiri kapena otsika kwambiri, sizingakhale bwino kuyambitsa kuyankha.Zomwe zili mu GC zoyambira kutsogolo ndi kumbuyo ziyenera kukhala pafupi ndi zomwezo kuti zipeze mtengo wofanana wa Tm ndi kutentha kwa annealing.
Mtengo wa Tm uyenera kukhala pakati pa 55-65 ° C momwe kungathekere, nthawi zambiri pafupifupi 60 ° C, ndipo mtengo wa Tm wa kumtunda ndi kumunsi kwa mtsinje uyenera kukhala woyandikira kwambiri, makamaka usapitirire 4 ° C.
4. Pewani kusankha A kumapeto kwa 3' koyambira
Pamene 3 ′ mapeto a choyambira sichikufanana, pali kusiyana kwakukulu mu kaphatikizidwe kabwino ka maziko osiyanasiyana.Pamene maziko omaliza ndi A, amathanso kuyambitsa kaphatikizidwe ka unyolo ngakhale pakakhala kusagwirizana, ndipo pamene maziko otsiriza ali T Pamene, mphamvu ya induction yolakwika imachepetsedwa kwambiri.Chifukwa chake, yesani kupewa kusankha A kumapeto kwa 3′ koyambira, ndipo ndi bwino kusankha T.
Ngati ndi probe primer, 5′ mapeto a kafukufuku sangakhale G, chifukwa ngakhale gulu limodzi la G litagwirizanitsidwa ndi gulu la olemba nkhani za fluorescent la FAM, G akhozanso kuzimitsa chizindikiro cha fulorosenti choperekedwa ndi gulu la FAM, zomwe zimabweretsa zotsatira zabodza.Kuwoneka.
5. Kugawa kwapansi
Kugawidwa kwa maziko anayi mu primer makamaka mwachisawawa, kupewa kupitilira 3 motsatizana G kapena C kumapeto kwa 3′, ndi kupitilira 3 motsatizana.G kapena C ndizosavuta kupanga ma pairing m'chigawo chotsatira cha GC.
6. Dera lopangira zoyambira liyenera kupewa zovuta zachiwiri.
Mapangidwe achiwiri opangidwa ndi chingwe chimodzi chazinthu zokulitsa chidzakhudza kupita patsogolo kwa PCR.Mwa kulosera ngati pali dongosolo lachiwiri muzotsatira zomwe mukufuna, yesetsani kupewa dera lino popanga zoyambira.
7. Zoyambira zokha komanso pakati pa zoyambira ziyenera kuyesetsa kupewa zoyambira zotsatizana.
Sipangakhale mgwirizano wotsatizana wa 4 pakati pa choyambira chokha ndi choyambira.Choyambira chokha sichiyenera kukhala ndi mndandanda wophatikizira, apo ayi imadzipinda kuti ipange mawonekedwe a hairpin, zomwe zingakhudze kuphatikiza kwa annealing kwa primer ndi template.
Zotsatizana zowonjezera sizingakhalepo pakati pa zoyambira zam'mwamba ndi zotsika.Kugwirizana pakati pa zoyambira kumatulutsa ma dimers oyambira, omwe amachepetsa magwiridwe antchito a PCR komanso kukhudza kulondola kwachulukidwe.Ngati mawonekedwe a primer-dimer ndi hairpin sangapeweke, mtengo wa △G usakhale wokwera kwambiri (uyenera kukhala wosakwana 4.5 kcal/mol).
8. Zoyambira zimakulitsa chinthu chomwe mukufuna.
Cholinga chachikulu cha kuzindikira kwa qPCR ndikumvetsetsa kuchuluka kwa jini yomwe mukufuna.Ngati kukulitsa kosakhazikika kumachitika, kuchuluka kwake kumakhala kolakwika.Chifukwa chake, zoyambira zitapangidwa, zimafunika kuyesedwa ndi BLAST, ndipo tsatanetsatane wazinthuzo zimafaniziridwa mumndandanda wazotsatira.
Kenaka, timatenga jini ya GAS6 yaumunthu (Kukula kumangidwa kwapadera kwa 6) monga chitsanzo kuti tipange zoyambira za qPCR.
01 funso jini
Homo GAS6kudzera ku NCBI.Apa, tiyenera kusamala kufananiza dzina la jini ndi zamoyo kuti zitsimikizire kuti zimagwirizana.
02 Pezani mndandanda wa jini
(1) Ngati ndondomeko ya DNA ya genomic, sankhani yoyamba, yomwe ndi ndondomeko ya DNA ya jini.
(2) Ngati ndondomeko ya chandamale ndi mRNA, sankhani yachiwiri.Mukalowa, dinani "CDS" mu tebulo ili m'munsimu.Mayendedwe amtundu wa bulauni ndi kalembedwe ka jini.
03 Zojambula zoyambira
Lowetsani mawonekedwe a Primer-BLAST
Lowetsani nambala yotsatizana ya jini kapena kutsatizana kwa Fasta kumtunda kumanzere, ndipo lembani magawo ofunikira.

Dinani "Pezani zoyambira" ndipo NCBI itulukira kuti ikuuzeni kuti kusankha kotereku kudzakulitsidwa kumitundu ina yolumikizira.Titha kuyang'ana mitundu yosiyanasiyana yolumikizirana ndikutumiza kuti tipeze zoyambira zoyenera (monga zikuwonekera pachithunzi pansipa).Izi zitha kutenga masekondi khumi kuti zichitike.
Kutentha kwapakati pazigawo zoyambazi ndi pafupifupi 60°C.Malinga ndi cholinga cha kuyesako, sankhani zoyambira zokhala ndi kutalika kwapakatikati, zodziwika bwino komanso kudzikwaniritsa pang'ono kwa zoyambira zoyeserera, ndipo kupambana kwake ndikokwera kwambiri!
04Chitsimikizo choyambirira
M'malo mwake, kuwonjezera pakupanga zoyambira, Primer-Blast imathanso kuwunika zoyambira zomwe tidazipanga tokha.Bwererani ku tsamba loyambira, lowetsani zoyambira zoyambira kumtunda ndi kumunsi zomwe tapanga, ndipo magawo ena sadzasinthidwa.Mukatumiza, mutha kuwona ngati zoyambira zilinso pamitundu ina.Ngati zonse zikuwonetsedwa pa jini yomwe tikufuna kukulitsa, kuwonetsa kuti tsatanetsatane wa zoyambira izi ndizabwino kwambiri!(Mwachitsanzo, izi ndi zotsatira zokha za funso loyamba!)

05 Chigamulo choyambirira cha khalidwe
Ndi mtundu wanji woyambira "wangwiro" womwe umaphatikizira "kukulitsa bwino mpaka mulingo", "mawonekedwe azinthu zokulitsa", ndi "zotsatira zodalirika zoyeserera"?
Kukulitsa luso

kusungunuka kopindika
Kuchita bwino kwa zoyambira kumafika 90% -110%, zomwe zikutanthauza kuti kukulitsa bwino ndikwabwino, ndipo nsonga yosungunuka imakhala ndi nsonga imodzi ndipo nthawi zambiri Tm> 80 ° C, zomwe zikutanthauza kuti kukulitsa kwake ndikwabwino.
 
Zogwirizana nazo:
Real Time PCR Easy-SYBR GREEN I
Real Time PCR Easy-Taqman