Ezivivinyweni ze-qPCR, idizayini yokuqala nayo iyisixhumanisi esibaluleke kakhulu.Ukuthi ama-primer afanelekile noma cha kuhlobene eduze nokuthi ukusebenza kahle kokukhulisa kufinyelela ezingeni, noma ngabe imikhiqizo ekhulisiwe icacile, kanye nokuthi imiphumela yokuhlola iyatholakala yini.
Ngakho-ke ungakwenza kanjani ukucaciswa kweprimer ye-qPCR kube ngcono?Ukusebenza kahle kwe-amplification ephezulu?
Namuhla, sizokuthatha ukuthi udizayine iziqalo ze-qPCR ndawonye, ​​futhi sivumele idizayini yokuqala ye-qPCR ibe ikhono elisebenzayo lomlando ekuhlolweni.
Lapho uklama ama-primers e-qPCR, ngokuvamile naka amaphuzu alandelayo: ama-primers kufanele aklanywe kuwo wonke ama-introns ngangokunokwenzeka, ubude bomkhiqizo kufanele bube ngu-100-300 bp, inani le-Tm kufanele libe eduze ngangokunokwenzeka ku-60°C, futhi iziqalo ezikhuphukayo nezingezansi komfula kufanele zibe seduze ngangokunokwenzeka, futhi isiphetho se-primer kufanele sibe ngu-G noma C, njll.
1. Idizayini yeziqalo ezihlanganisa izithulo
Lapho uklama ama-primers e-qPCR, ukukhetha ama-primer aklanywe kuwo wonke ama-intron kungavimbela ithempulethi ye-gDNA ukuthi ingakhuliswa, futhi imikhiqizo yonke ithathwe ekwandiseni i-cDNA, ngaleyo ndlela kuqede umthelela wokungcola kwe-gDNA.
2. Ubude bokuqala
Ubude be-primer ngokuvamile buphakathi kuka-18-30 nt, futhi ubude bomkhiqizo wokukhuliswa kufanele bulawuleke phakathi kuka-100-300 bp kakhulu ngangokunokwenzeka.
Uma i-primer imfushane kakhulu, izoholela ekukhuliseni okungaqondile, futhi uma iyinde kakhulu, izokwakha kalula isakhiwo sesibili (njengesakhiwo se-hairpin).Uma umkhiqizo wokukhulisa umude kakhulu, awufanele ukusabela kwe-polymerase, okuzothinta ukusebenza kahle kokukhulisa i-PCR.
3. Okuqukethwe kwe-GC kanye nenani le-Tm
Okuqukethwe kwe-GC kweziqalo kufanele kulawulwe phakathi kuka-40% no-60%.Uma iphezulu kakhulu noma iphansi kakhulu, akusizi ukuqalisa ukusabela.Okuqukethwe kwe-GC kweziqalisi eziya phambili nangemuva kufanele kusondele okufanayo ukuze kutholwe inani elifanayo le-Tm kanye nezinga lokushisa lokudonsa.
Inani le-Tm kufanele libe phakathi kuka-55-65°C ngangokunokwenzeka, ngokuvamile libe ngu-60°C, futhi inani le-Tm lasenhla nomfula kufanele libe seduze ngangokunokwenzeka, okungcono lingabi ngaphezu kuka-4°C.
4. Gwema ukukhetha u-A ekupheleni kuka-3′ kwesiqalo
Uma isiphetho esingu-3′ se-primer singafani, kunomehluko omkhulu ekusebenzeni kahle kokuhlanganiswa kwezisekelo ezihlukene.Uma isisekelo sokugcina singu-A, singakwazi futhi ukuqalisa ukuhlanganiswa kweketango ngisho nalapho isisekelo sokugcina singu-T Lapho, ukusebenza kahle kokungeniswa okungafani kuncipha kakhulu.Ngakho-ke, zama ukugwema ukukhetha u-A ekugcineni kwe-3′, futhi kungcono ukhethe i-T.
Uma kuyi-probe primer, isiphetho esingu-5′ se-probe asinakuba ngu-G, ngoba ngisho noma isisekelo se-G esisodwa sixhunywe eqenjini lentatheli ye-FAM fluorescent, u-G angaphinda acime isignali ye-fluorescent ekhishwa iqembu le-FAM, okuholela emiphumeleni engemihle engamanga.Vele.
5. Ukusabalalisa okuyisisekelo
Ukusatshalaliswa kwezisekelo ezine ku-primer kukhethwa okungahleliwe, kugwema okungaphezulu koku-3 okulandelanayo kwe-G noma u-C ekugcineni kuka-3′, nangaphezu kuka-3 ngokulandelana.I-G noma i-C kulula ukukhiqiza ukumatanisa endaweni yokulandelana ecebile nge-GC.
6. Isifunda sokuklama i-primer kufanele sigweme izakhiwo zesibili eziyinkimbinkimbi.
Isakhiwo sesibili esakhiwe umucu owodwa womkhiqizo wokukhuliswa sizothinta ukuqhubeka kahle kwe-PCR.Ngokubikezela ukuthi kukhona yini ukwakheka kwesibili ngokulandelana okuhlosiwe kusengaphambili, zama ukugwema lesi sifunda ekwakhiweni kwama-primers.
7. Ama-primer ngokwawo naphakathi kwama-primers kufanele azame ukugwema izisekelo ezilandelanayo ezilandelanayo.
Angeke kube khona ukuphelelisana kwesisekelo okungu-4 okulandelanayo phakathi kwe-primer ngokwayo kanye ne-primer.I-primer ngokwayo akufanele ibe nokulandelana okuhambisanayo, ngaphandle kwalokho izozigoqa ukuze yakhe isakhiwo se-hairpin, esizothinta inhlanganisela ye-anneal ye-primer kanye nesifanekiso.
Ukulandelana okuhambisanayo akukwazi ukuba khona phakathi kweziqalo zaphezulu nomfula.Ukuhambisana phakathi kwama-primers kuzokhiqiza ama-primer dimers, azonciphisa ukusebenza kahle kwe-PCR futhi athinte nokunemba komthamo.Uma i-primer-dimer nezakhiwo ze-hairpin zingagwemeki, inani △G akumele libe phezulu kakhulu (kufanele libe ngaphansi kuka-4.5 kcal/mol).
8. Ama-primers akhulisa umkhiqizo othile oqondiwe.
Umgomo omkhulu wokutholwa kwe-qPCR ukuqonda ukuchichima kofuzo oluqondiwe.Uma ukukhuliswa okungaqondile kwenzeka, ukulinganisa kuzoba okungalungile.Ngakho-ke, ngemuva kokuthi ama-primer eklanyelwe, adinga ukuhlolwa yi-BLAST, futhi ukucaciswa kwemikhiqizo kuqhathaniswa kudathabhesi yokulandelana.
Okulandelayo, sithatha isakhi sofuzo somuntu se-GAS6 (Ukukhula kokubopha okuqondile okungu-6) njengesibonelo sokudizayina iziqalo ze-qPCR.
01 umbuzo wofuzo
I-Homo GAS6nge-NCBI.Lapha, kufanele sinake ukuqhathanisa igama lofuzo nezinhlobo zofuzo ukuze siqinisekise ukuthi ziyahambisana.
02 Thola ukulandelana kofuzo
(1) Uma ukulandelana okuhlosiwe kuyi-DNA ye-genomic, khetha eyokuqala, okuwukulandelana kwe-DNA ye-genomic yofuzo.
(2) Uma ukulandelana okuqondiwe kuyi-mRNA, khetha okwesibili.Ngemva kokufaka, chofoza u-“CDS” kuthebula elingezansi.Ukulandelana kwangemuva okunsundu wukulandelana kwekhodi kofuzo.
03 Iziqalo zedizayini
Faka isixhumi esibonakalayo se-Primer-BLAST
Faka inombolo yokulandelana kofuzo noma ukulandelana ngefomethi ye-Fasta ngenhla kwesokunxele, bese ugcwalisa imingcele efanele.

Chofoza okuthi “Thola ama-primers” futhi i-NCBI izovela ukuze ikutshele ukuthi ukukhetha okunjalo kwepharamitha kuzokhuliswa kwezinye izinhlobo zokuhlanganisa.Singabheka okuhlukile kokuhlanganisa okuhlukile futhi sikuthumele ukuze sithole ukupheya okufanelekile (njengoba kukhonjisiwe esithombeni esingezansi).Le nqubo ingase ithathe amashumi amasekhondi ukusebenza.
Amazinga okushisa e-annealing alawa mapheya okuqala azungeze u-60°C.Ngokwenhloso yokuhlolwa, khetha ama-primer anobude obumaphakathi, ukucaciswa okuhle kanye nokuzigcwalisa okuncane kweziqalo zokuhlola, futhi izinga lempumelelo liphezulu impela!
04Ukuqinisekiswa kokucaciswa kokuqala
Eqinisweni, ngaphezu kokuklama ama-primer, i-Primer-Blast ingaphinda ihlole iziqalo esiziklame ngokwethu.Buyela ekhasini le-primer design, faka ama-primers angenhla naphansi omfula esiwaklamile, futhi amanye amapharamitha ngeke alungiswe.Ngemva kokuhambisa, ungabona ukuthi ingabe ama-primer akhona yini kwezinye izakhi zofuzo.Uma zonke ziboniswa kufuzo esifuna ukulikhulisa, okubonisa ukuthi ukucaciswa kwaleli pheya leziqalo kuhle kakhulu!(Isibonelo, lona ukuphela komphumela wombuzo wokuqala!)

05 Ukwahlulela kwekhwalithi yokuqala
Hlobo luni lwe-primer i-primer "ephelele" ehlanganisa "ukusebenza kahle kokukhulisa kuze kufike ezingeni elijwayelekile", "izici zomkhiqizo okhulisiwe", kanye "nemiphumela yokuhlola ethembekile"?
Ukusebenza kahle kokukhulisa

ijika elincibilikayo
Ukusebenza kahle kokukhulisa ama-primers kufinyelela ku-90% -110%, okusho ukuthi ukusebenza kahle kokukhulisa kuhle, futhi ijika lokuncibilika linokuphakama okukodwa futhi ngokuvamile i-Tm>80°C, okusho ukuthi ukucaciswa kokukhulisa kuhle.
 
Imikhiqizo Ehlobene:
Isikhathi Sangempela PCR Easy–SYBR GREEN I
Isikhathi Sangempela PCR Easy-Taqman