Ibintu bishobora gutera ingaruka ku mikorere ya PCR

Mu gihe cyo gukora PCR reaction, hari ibintu bimwe na bimwe bikunze kugaragara.
Bitewe n’uko PCR ishobora kwanduza abantu benshi, ubwandu bufatwa nk’ikintu cy’ingenzi bigira ingaruka ku bisubizo bya PCR kandi bushobora gutanga ibisubizo bitari byo.
Ikindi kintu cy’ingenzi ni amasoko atandukanye atuma habaho ibisubizo bitari byo. Iyo igice kimwe cyangwa byinshi by’ingenzi by’uruvange rwa PCR cyangwa igisubizo cyo kongera imbaraga ubwabyo bikumiriwe cyangwa bivangiwe, isuzuma ryo gusuzuma rishobora kubangamira. Ibi bishobora gutuma umusaruro ugabanuka ndetse n’ibitari byo.
Uretse gukumira, gutakaza ubusugire bwa aside nucleic fatizo bishobora guterwa n’uburyo bwo kohereza no/cyangwa uburyo bwo kubika mbere yo gutegura icyitegererezo. By’umwihariko, ubushyuhe bwinshi cyangwa uburyo budahagije bwo kubika bishobora kwangiza uturemangingo na aside nucleic. Gufata uturemangingo n’uturemangingo no gushyira parafini mu mubiri ni impamvu zizwi cyane zitera gucikamo ibice bya ADN n’ikibazo gihoraho (reba Ishusho ya 1 na 2). Muri ibi bihe, no kwitandukanya no gutunganya neza ntacyo bizafasha.

Ishusho ya 1 | Ingaruka zo kudakora neza kw'ingirabuzimafatizo ku buziranenge bwa ADN
Agarose gel electrophoresis yagaragaje ko ubwiza bwa ADN yakuwe mu bice bya parafini byo gusuzuma umurambo bwari butandukanye cyane. ADN y'uburebure butandukanye bw'ibice byayo yabonekaga mu bicuruzwa bitewe n'uburyo bwo kuyishyiraho. ADN yabikwaga gusa iyo yashyizwe mu bipimo bya kera byakonjeshejwe no muri formalin ya neutral yabugenewe. Gukoresha formalin ya Bouin ishaje cyane cyangwa idafite aside ya formike byateje igihombo gikomeye cya ADN. Igice gisigaye cyagabanutse cyane.
Ibumoso, uburebure bw'ibice bugaragazwa mu ma pair ya kilobase (kbp)

Ishusho ya 2 | Gutakaza ubusugire bw'ibipimo bya aside nucleic
(a) Icyuho cya 3′-5′ ku migozi yombi kizatera icyuho muri ADN y’ingenzi. Ikorwa rya ADN rizakomeza kubaho ku gice gito. Ariko, iyo ahantu hanini habuze agace ka ADN, haboneka gusa uburyo bwo kongera ingufu. Mu gihe cyiza cyane, ibice bishobora kongera kuzura, ariko umusaruro uzaba muto kandi uri munsi y’urwego rwo kubimenya.
(b) Gutakaza bases, ahanini bitewe no gukurwaho kw'isuku no kwirema kwa thymidine dimer, bituma umubare wa H-bonds ugabanuka ndetse na Tm ikagabanuka. Mu gihe cyo gushyuha cyane, primers zizashonga zikava kuri ADN ya matrix kandi ntizizongera gushonga nubwo haba hari ibintu bitoroshye.
(c) Ishingiro rya thymine ryegeranye rikora TT dimer.
Ikindi kibazo gikunze kugaragara mu gusuzuma molekile ni ukurekurwa guke kwa aside nucleic target ugereranije no gukuramo phenol-chloroform. Mu bihe bikomeye, ibi bishobora kujyana n'ibintu bitari byo. Igihe kinini gishobora kurokorwa no guteka lysis cyangwa igogorwa rya enzymatic ry'ibisigazwa by'uturemangingo, ariko ubu buryo bukunze gutuma PCR idakora neza bitewe no kutarekurwa guke kwa aside nucleic.

Kubuza imikorere ya polymerase mu gihe cyo kongera imbaraga

Muri rusange, gukumira bikoreshwa nk'igitekerezo cy'ibikoresho kugira ngo hasobanurwe ibintu byose bituma habaho ibisubizo bya PCR bitari byiza. Mu buryo bwa biochemical gusa, gukumira bigarukira ku bikorwa bya enzyme, ni ukuvuga, bigabanya cyangwa bikabuza guhindura substrate-product binyuze mu gukorana n'ahantu hakora ha polymerase ya ADN cyangwa cofactor yayo (urugero, Mg2+ kuri Taq DNA polymerase).
Ibice biri mu gipimo cyangwa mu buryo butandukanye bwo gufunga no gusohora ibintu birimo reagents bishobora kubuza enzyme cyangwa gufata cofactors zayo (urugero: EDTA), bityo bigatuma polymerase idakora, bityo bigatuma ibisubizo bya PCR bigabanuka cyangwa bitari byo.
Ariko, imikoranire myinshi hagati y’ibice bigize reaction na aside nucleic zirimo intego nabyo byitwa 'PCR inhibitors'. Iyo ubusugire bw’akaremangingo buhungabanyijwe no kwitandukanya maze aside nucleic ikarekurwa, imikoranire hagati y’icyitegererezo n’umuti ukikije hamwe n’igice gikomeye ishobora kubaho. Urugero, 'abacana' bashobora gufata ADN imwe cyangwa ebyiri binyuze mu mikoranire idahuje n’ingirabuzimafatizo kandi bakabangamira kwitandukanya no gusukura binyuze mu kugabanya umubare w’ibice bigera ku muyoboro wa PCR reaction.
Muri rusange, imiti igabanya ubukana bwa PCR iboneka mu mazi menshi yo mu mubiri no mu bipimo bikoreshwa mu gusuzuma indwara (urea mu nkari, hemoglobin na heparin mu maraso), inyongeramusaruro (ibice by’ibinyabuzima, glycogen, ibinure, iyoni za Ca2+) n’ibice biri mu bidukikije (phenols, ibyuma biremereye)

Uburyo bwo gukumira PCR bukunze kugaragara buboneka mu dukoko twa bagiteri na eukaryotic, ADN idashingiye ku ntego, macromolecules zifata ADN z’uturemangingo tw’umubiri ndetse n’ibikoresho bya laboratwari nka gants na pulasitiki. Gusukura aside nucleic mu gihe cyo gukuramo cyangwa nyuma yo gukuramo ni bwo buryo bwiza bwo gukuraho PCR ibuza.
Muri iki gihe, ibikoresho bitandukanye byo gukuramo ibintu byikora bishobora gusimbura protocole nyinshi zikoreshwa n'intoki, ariko kugarura no gusukura intego 100% ntibyigeze bigerwaho. Ibishobora gutuma ibintu bidashobora gukoreshwa bishobora kuba bikiri muri aside nucleic isukuye cyangwa bikaba byaratangiye gukoreshwa. Hari ingamba zitandukanye zo kugabanya ingaruka z'ibishobora gukoreshwa. Guhitamo polymerase ikwiye bishobora kugira ingaruka zikomeye ku bikorwa byo gukumira. Ubundi buryo bwagaragaye bwo kugabanya gukumira PCR ni ukongera ubwinshi bwa polymerase cyangwa gukoresha inyongera nka BSA.
Kubuza ibikorwa bya PCR bishobora kugaragazwa no gukoresha uburyo bwo kugenzura ubuziranenge bw'imikorere y'umubiri (IPC).
Hagomba kwitonderwa gukuraho ibintu byose bigabanya ubushyuhe n'ibindi bisubizo biri mu gikoresho cyo gukuramo, nka ethanol, EDTA, CETAB, LiCl, GuSCN, SDS, isopropanol na phenol, mu gice cy'aside nucleic isolate hakoreshejwe intambwe yo gukaraba neza. Bitewe n'ingano yabyo, bishobora gukora cyangwa kubuza PCR.