Abubuwan tsoma baki a cikin halayen PCR

Yayin da PCR ke yi, ana yawan fuskantar wasu abubuwan da ke sa baki.
Saboda tsananin girman hankali na PCR, ana ɗaukar gurɓatawa a matsayin ɗaya daga cikin mahimman abubuwan da ke shafar sakamakon PCR kuma yana iya haifar da sakamako mai kyau na ƙarya.
Hakanan mahimmin mahimmanci shine tushe iri-iri waɗanda ke haifar da sakamako mara kyau.Idan ɗayan ko fiye da mahimman sassa na cakuda PCR ko haɓakawa kanta an hana su ko tsoma baki tare da su, za a iya hana gwajin ganowa.Wannan na iya haifar da raguwar inganci har ma da sakamako mara kyau na ƙarya.
Baya ga hanawa, asarar ƙimar ƙimar nucleic acid mai niyya na iya faruwa saboda jigilar kaya da / ko yanayin ajiya kafin shirya samfurin.Musamman yanayin zafi ko rashin isasshen ajiya na iya haifar da lalacewar sel da acid nucleic.Gyaran tantanin halitta da nama da saka paraffin sune sanannun abubuwan da ke haifar da rarrabuwar DNA da matsala mai tsayi (duba Figures 1 da 2).A cikin waɗannan lokuta, ko da mafi kyawun keɓewa da tsarkakewa ba zai taimaka ba.

Hoto 1 |Tasirin immobilization akan amincin DNA
Agarose gel electrophoresis ya nuna cewa ingancin DNA da ke ware daga sassan paraffin na autopsies ya bambanta sosai.DNA na matsakaicin tsayin guntu daban-daban ya kasance a cikin abubuwan da aka cire dangane da hanyar gyarawa.An adana DNA ne kawai lokacin da aka gyara shi a cikin samfuran daskararre na asali kuma a cikin tsari mai tsaka tsaki.Amfani da ƙaƙƙarfan acidic Bouin fixative ko unbuffered, formic acid mai ɗauke da formalin ya haifar da babbar asarar DNA.Ragowar juzu'in ya rabu sosai.
A gefen hagu, ana bayyana tsawon gutsure a cikin nau'i-nau'i na kilobase (kbp)

Hoto 2 |Rashin mutuncin makasudin acid nucleic
(a) Rata ta 3'-5' akan igiyoyin biyu zai haifar da hutu a cikin DNA da aka yi niyya.kira na DNA har yanzu zai faru akan ƙaramin guntu.Duk da haka, idan an rasa wurin da ake cirewa na farko akan guntun DNA, haɓakar layin layi kawai yana faruwa.A cikin yanayin da ya fi dacewa, gutsuttsura na iya sake dawo da juna, amma abin da ake samu zai zama ƙanana kuma ƙasa da matakan ganowa.
(b) Rashin tushe, galibi saboda depurination da samuwar thymidine dimer, yana haifar da raguwar adadin H-bonds da raguwa a cikin Tm.A lokacin lokacin ɗumamar elongated, firam ɗin za su narke daga matrix DNA kuma ba za su ɓata ba ko da ƙarƙashin ƙarancin yanayi.
(c) Matsalolin thymine da ke kusa suna samar da dimer TT.
Wata matsalar gama gari wacce sau da yawa ke faruwa a cikin bincike na ƙwayoyin cuta ita ce ƙarancin sakin acid nucleic da aka yi niyya idan aka kwatanta da hakar phenol-chloroform.A cikin matsanancin yanayi, ana iya haɗa wannan tare da rashin ƙarfi na ƙarya.Ana iya adana lokaci mai yawa ta hanyar tafasa lysis ko narkewar enzymatic na tarkacen tantanin halitta, amma wannan hanyar sau da yawa yana haifar da ƙarancin fahimtar PCR saboda ƙarancin sakin nucleic acid.

Hana ayyukan polymerase yayin haɓakawa

Gabaɗaya, ana amfani da hanawa azaman ra'ayi na kwantena don bayyana duk abubuwan da ke haifar da mafi kyawun sakamakon PCR.A cikin ma'anar sinadarai mai mahimmanci, hanawa yana iyakance ga ayyukan enzyme, watau yana ragewa ko hana jujjuyawar samfur ta hanyar hulɗa tare da wurin aiki na DNA polymerase ko mai haɗin gwiwa (misali, Mg2+ na Taq DNA polymerase).
Abubuwan da aka haɗa a cikin samfurin ko daban-daban buffers da tsantsa mai ɗauke da reagents na iya hana enzyme kai tsaye ko kama masu haɗin gwiwa (misali EDTA), ta haka yana kunna polymerase kuma hakan yana haifar da raguwar ko ƙarancin sakamakon PCR.
Koyaya, yawancin mu'amala tsakanin abubuwan amsawa da kuma acid nucleic masu ɗauke da manufa suma an sanya su azaman 'masu hana PCR'.Da zarar an rushe mutuncin tantanin halitta ta hanyar keɓewa kuma aka saki acid nucleic, hulɗar tsakanin samfurin da maganin da ke kewaye da shi da kuma lokaci mai ƙarfi na iya faruwa.Misali, 'masu zamba' na iya daure DNA guda-ko-biyu ta hanyar mu'amalar da ba ta dace ba kuma suna tsoma baki tare da keɓewa da tsarkakewa ta hanyar rage adadin maƙasudi waɗanda a ƙarshe suka isa jirgin ruwan PCR.
Gabaɗaya, masu hana PCR suna kasancewa a cikin yawancin ruwaye na jiki da reagents da ake amfani da su don gwaje-gwajen bincike na asibiti (urea a cikin fitsari, haemoglobin da heparin a cikin jini), abubuwan abinci mai gina jiki (haɓaka kwayoyin halitta, glycogen, mai, Ca2+ ions) da abubuwan da ke cikin muhalli (phenols). , karafa masu nauyi)

Ana iya samun ƙarin inhibitors na PCR a cikin ƙwayoyin cuta da ƙwayoyin eukaryotic, DNA marasa manufa, DNA-daure macromolecules na matrices nama da kayan aikin dakin gwaje-gwaje kamar safar hannu da robobi.Tsaftace acid nucleic lokacin ko bayan hakar shine hanyar da aka fi so don cire masu hana PCR.
A yau, kayan aikin hakar mai sarrafa kansa daban-daban na iya maye gurbin ka'idoji na hannu da yawa, amma 100% farfadowa da/ko tsarkake maƙasudi ba a taɓa samun nasara ba.Ƙila masu hana masu hanawa har yanzu suna kasancewa a cikin tsarkakakkun acid nucleic ko kuma sun riga sun yi tasiri.Akwai dabaru daban-daban don rage tasirin masu hanawa.Zaɓin zaɓi na polymerase mai dacewa zai iya samun tasiri mai mahimmanci akan ayyukan hanawa.Sauran hanyoyin da aka tabbatar don rage hanawar PCR suna ƙara yawan tattarawar polymerase ko amfani da ƙari kamar BSA.
Ana iya nuna hana halayen PCR ta hanyar amfani da sarrafa ingancin tsari na ciki (IPC).
Dole ne a kula don cire duk reagents da sauran mafita a cikin kayan hakar, irin su ethanol, EDTA, CETAB, LiCl, GuSCN, SDS, isopropanol da phenol, daga keɓewar acid nucleic ta hanyar wankewa sosai.Dangane da maida hankalinsu, za su iya kunna ko hana PCR.