Kwimifuniselo ye-qPCR, uyilo lweprimer ikwalikhonkco elibaluleke kakhulu.Ingaba i-primers ifanelekile okanye ayifanelekanga inxulumene ngokusondeleyo nokuba impumelelo yokukhulisa ifikelela kumgangatho, ingaba iimveliso ezikhulisiwe zichanekile, kunye nokuba iziphumo zovavanyo ziyafumaneka.
Ke ungayenza njani i-qPCR primer icace ngcono?Ukusebenza kokwandisa okuphezulu?
Namhlanje, siya kukuthatha ukuba uyile iiprimers ze-qPCR kunye, kwaye uvumele uyilo lweprimer ye-qPCR lube sisakhono esisebenzayo kwimifuniselo.
Xa uqulunqa ii-primers ze-qPCR, ngokuqhelekileyo ubeke ingqalelo kula manqaku alandelayo: ii-primers kufuneka zenziwe kwii-introns kangangoko kunokwenzeka, ubude bemveliso kufuneka bube yi-100-300 bp, ixabiso le-Tm kufuneka libe lisondele kangangoko kunokwenzeka kwi-60 ° C, kunye ne-primers ephezulu kunye ne-downstream kufuneka ibe kufutshane kangangoko kunokwenzeka, kwaye isiphelo se-primer kufuneka sibe yi-G okanye i-C, njl.
1. Uyilo lwee-primers ezithatha ii-introns
Xa kuyilwa iiprimers ze-qPCR, ukukhetha iiprimers eziyilwe kuzo zonke ii-introns kunokuthintela ithemplate ye-gDNA ekubeni yandiswe, kwaye iimveliso zonke zithathwe kukwandiswa kwe-cDNA, ngaloo ndlela kucinywa impembelelo yongcoliseko lwe-gDNA.
2. Ubude bePrimer
Ubude be-primer buphakathi kwe-18-30 nt, kwaye ubude bemveliso yokukhulisa kufuneka ilawulwe phakathi kwe-100-300 bp kangangoko kunokwenzeka.
Ukuba i-primer imfutshane kakhulu, iya kukhokelela ekukhuliseni okungaqhelekanga, kwaye ukuba ide kakhulu, iya kwenza ngokulula isakhiwo sesibini (njengesakhiwo se-hairpin).Ukuba imveliso yokukhulisa inde kakhulu, ayifanelekanga ukusabela kwepolymerase, eya kuchaphazela ukusebenza kakuhle kwe-PCR yokukhulisa.
3. Umxholo we-GC kunye nexabiso le-Tm
Umxholo we-GC weeprimers kufuneka ulawulwe phakathi kwe-40% kunye ne-60%.Ukuba iphezulu kakhulu okanye iphantsi kakhulu, ayikuncedi ukuqalisa ukusabela.Umxholo we-GC we-primers ngaphambili kunye ne-reverse primers kufuneka usondele ngokufanayo ukuze ufumane ixabiso elifanayo le-Tm kunye nobushushu bokutshisa.
Ixabiso le-Tm kufuneka libe phakathi kwe-55-65 ° C ngokusemandleni, ngokubanzi malunga ne-60 ° C, kwaye ixabiso le-Tm lokunyuka kunye nezantsi kufuneka libe kufutshane kangangoko kunokwenzeka, ngokukhethekileyo lingabi ngaphezu kwe-4 ° C.
4. Kuphephe ukukhetha uA kwi-3′ ekupheleni kweprimer
Xa isiphelo se-3 se-primer singahambelani, kukho ukungafani okukhulu kwi-synthesis ye-synthesis yeziseko ezahlukeneyo.Xa isiseko sokugqibela singu-A, sinokuphinda siqalise i-chain synthesis nakwimeko yokungahambi kakuhle, kwaye xa isiseko sokugqibela singu-T Xa, ukusebenza kakuhle kokungeniswa kwe-mismatch kuncitshiswe kakhulu.Ngoko ke, zama ukunqanda ukukhetha u-A ekupheleni kwe-3 ye-primer, kwaye kungcono ukhethe i-T.
Ukuba i-probe primer, i-5 'isiphelo se-probe ayikwazi ukuba yi-G, kuba nangona isiseko esisodwa se-G sixhunywe kwiqela le-FAM ye-fluorescent reporter, i-G inokuphinda icime isignali ye-fluorescent ekhutshwe liqela le-FAM, okubangela iziphumo ezingalunganga.Vela.
5. Ukuhanjiswa kwesiseko
Ukusasazwa kweziseko ezine kwi-primer kukhethwa ukuba i-random, ukuphepha ngaphezu kwe-3 elandelelanayo ye-G okanye i-C ekupheleni kwe-3, kwaye ngaphezu kwe-3 ngokulandelelana.I-G okanye i-C kulula ukuvelisa ukubhanqa kwingingqi yokulandelelana kwe-GC-rich.
6. Ummandla woyilo lwe-primer kufuneka ugweme izakhiwo eziyinkimbinkimbi zesibini.
Isakhiwo sesibini esenziwe ngumcu omnye wemveliso yokukhulisa iya kuchaphazela inkqubela phambili yePCR.Ngokuqikelela ukuba ngaba kukho isakhiwo sesibini kulandelelwano olujoliswe kuyo kwangaphambili, zama ukuphepha lo mmandla ekuyilweni kwee-primers.
7. Ii-primers ngokwazo kunye naphakathi kwee-primers kufuneka zizame ukuphepha iziseko ezihambelanayo ezilandelelanayo.
Akunakubakho ukulandelana kwesiseko esi-4 esilandelelanayo phakathi kwe-primer ngokwayo kunye ne-primer.I-primer ngokwayo akufanele ibe nolandelelwano oluncedisayo, ngaphandle koko iya kuzigoqa ukuze yenze isakhiwo se-hairpin, esiya kuchaphazela ukudibanisa kwe-annealing ye-primer kunye ne-template.
Ulandelelwano olongezelelweyo alukwazi ukubakho phakathi kweeprayimari ezinyukayo nezisezantsi.Ukuhambelana phakathi kwe-primers kuya kuvelisa i-primer dimers, eya kunciphisa ukusebenza kwe-PCR kwaye ichaphazele ukuchaneka kobungakanani.Ukuba i-primer-dimer kunye ne-hairpin structures azinakuthintelwa, ixabiso △G akufunekanga libe phezulu kakhulu (kufuneka libe ngaphantsi kwe-4.5 kcal/mol).
8. Iiprimers zandisa imveliso ethile ekujoliswe kuyo.
Eyona njongo iphambili yokubhaqwa kwe-qPCR kukuqonda ubuninzi bejini ekujoliswe kuyo.Ukuba i-amplification engekho ngqo iyenzeka, ubungakanani bexabiso luya kuba lungachanekanga.Ngoko ke, emva kokuba ii-primers ziyilwe, kufuneka zivavanywe yi-BLAST, kwaye ukucaciswa kweemveliso kuthelekiswa nedatha yokulandelelana.
Okulandelayo, sithatha i-GAS6 yomntu (Ukukhula ukubanjwa kwe-6) gene njengomzekelo wokuyila iiprimers ze-qPCR.
01 umbuzo gene
Homo GAS6ngokusebenzisa i-NCBI.Apha, kufuneka sinikele ingqalelo ekuthelekiseni igama lemfuza kunye neentlobo ukuqinisekisa ukuba ziyahambelana.
02 Fumana ulandelelwano lwemfuza
(1) Ukuba ulandelelwano olujoliswe kuyo luyi-DNA ye-genomic, khetha eyokuqala, eyinkqubo ye-DNA ye-genomic ye-gene.
(2) Ukuba ulandelelwano olujoliswe kuyo yi-mRNA, khetha eyesibini.Emva kokungena, cofa u-“CDS” kwitheyibhile engezantsi.Ulandelelwano olumdaka ngasemva lulandelelwano lwekhowudi yejini.
03 Yila iiprimers
Ngenisa i-Primer-BLAST interface
Faka inombolo yolandelelwano lwemfuza okanye ulandelelwano kwifomathi ye-Fasta ngasentla ngasekhohlo, kwaye ugcwalise iiparamitha ezifanelekileyo.

Cofa u-"Fumana iiprimers" kwaye i-NCBI iya kuvela ukuze ikuxelele ukuba ukhetho olunjalo lweparamitha luya kwandiswa kwezinye izinto ezahlukeneyo zokuhlanganisa.Sinokujonga iintlobo ezahlukeneyo zokudibanisa kwaye sizithumele ukuze sifumane i-primer pair efanelekileyo (njengoko kubonisiwe kumfanekiso ongezantsi).Le nkqubo inokuthatha amashumi emizuzwana ukuqalisa.
Amaqondo obushushu asezantsi kwezi peri zeprimer ajikeleze 60°C.Ngokwenjongo yovavanyo, khetha iiprimers ezinobude obuphakathi, ukuchaneka okulungileyo kunye nokuzalisa okungaphantsi kokwenza iiprimer zovavanyo, kwaye izinga lempumelelo liphezulu kakhulu!
04Isiqinisekiso sokuqala esicacileyo
Ngapha koko, ukongeza ekuyileni iiprimers, iPrimer-Blast inokuphinda ivavanye iiprimers esiziyileyo ngokwethu.Buyela kwiphepha loyilo lwe-primer, faka i-primers ephezulu kunye nesezantsi esiyiyile, kunye nezinye iiparameters aziyi kulungelelaniswa.Emva kokuthumela, unokubona ukuba ipere ye-primers zikhona na kwezinye iijini.Ukuba zonke ziboniswa kwi-gene esifuna ukuyikhulisa, ebonisa ukuba ukucaciswa kwesi sibini se-primers kukhulu!(Umzekelo, esi kuphela kwesiphumo sombuzo wokuqala!)

05 Isigwebo somgangatho wokuqala
Loluphi uhlobo lwe-primer oluyi-primer "egqibeleleyo" edibanisa "ubuchule bokukhulisa ukuya kumgangatho", "iimpawu zemveliso ezikhulisiwe", kunye "neziphumo zovavanyo ezithembekileyo"?
Ukwandiswa kobuchule

ijika elinyibilikayo
Ubuchule bokukhulisa i-primers bufikelela kwi-90% -110%, oku kuthetha ukuba ukunyuswa kwe-amplification kulungile, kwaye i-curve yokunyibilika inencopho eyodwa kwaye ngokuqhelekileyo i-Tm> 80 ° C, oku kuthetha ukuba ukucaciswa kwe-amplification kulungile.
 
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