Impamvu zo kwivanga mubitekerezo bya PCR

Mugihe cya PCR, ibintu bimwe bivanga bikunze guhura nabyo.
Bitewe no kumva cyane PCR, kwanduza bifatwa nkimwe mubintu byingenzi bigira ingaruka kubisubizo bya PCR kandi bishobora gutanga ibisubizo byiza bitari byo.
Kunegura kimwe nisoko itandukanye iganisha kubisubizo-bibi. Niba igice kimwe cyangwa byinshi byingenzi bivanze na PCR cyangwa reaction ya amplification ubwayo irahagaritswe cyangwa ikabangamirwa, isuzuma ryo gusuzuma rishobora kubangamirwa. Ibi birashobora gutuma imikorere igabanuka ndetse nibisubizo bibi.
Usibye kubuza, gutakaza intego ya nucleic acide irashobora kubaho bitewe no kohereza hamwe / cyangwa ububiko mbere yo gutegura icyitegererezo. By'umwihariko, ubushyuhe bwinshi cyangwa ububiko budahagije bushobora gutera kwangirika kwa selile na acide nucleic. Gutunganya ingirabuzimafatizo hamwe nuduce hamwe no gushyiramo paraffine ni bizwi neza ko bitera ADN gucikamo ibice hamwe nikibazo gihoraho (reba Ishusho 1 na 2). Muri ibi bihe, ndetse no kwigunga no kwezwa ntabwo bizafasha.

Igishushanyo 1 | Ingaruka zo kudahinduka ku busugire bwa ADN
Agarose gel electrophorei yerekanaga ko ubwiza bwa ADN bwitaruye ibice bya paraffin ya autopsie bitandukanye cyane. ADN yuburebure butandukanye buringaniye yari ihari mugukuramo bitewe nuburyo bwo gukosora. ADN yabitswe gusa mugihe yashizwemo icyitegererezo cyakonjeshejwe kavukire no muri formine itabogamye. Gukoresha aside irike cyane ya Bouin ikosora cyangwa idakumiriwe, aside irike irimo formine yatumye ADN itakaza cyane. Igice gisigaye cyacitsemo ibice.
Ibumoso, uburebure bwibice bugaragarira muri kilobase ebyiri (kbp)

Igishushanyo 2 | Gutakaza ubunyangamugayo bwa acide nucleic
(a) Ikinyuranyo cya 3′-5 ′ kumirongo yombi bizavamo gucamo ADN intego. synthesis ya ADN izakomeza kugaragara ku gice gito. Ariko, niba urubuga rwa primer annealing rwabuze ku gice cya ADN, gusa umurongo wo kwongera umurongo ubaho. Mugihe cyiza cyane, ibice bishobora gusubiranamo, ariko umusaruro uzaba muto kandi munsi yurwego rwo gutahura.
. Mugihe cyigihe kirekire cyo gushyuha, primers izashonga kure ya matrike ya ADN kandi ntishobora gukomera nubwo haba mubihe bitoroshye.
(c) Ibishingwe bya thymine byegeranye bigize TT dimer.
Ikindi kibazo gikunze kugaragara mugupima molekuline ni ukurekura-kutarekura neza ya acide nucleic acide ugereranije no gukuramo fenol-chloroform. Mubihe bikabije, ibi birashobora guhuzwa nibibi bibi. Umwanya munini urashobora gukizwa no guteka lysis cyangwa igogorwa ryimisemburo yimyanda ya selile, ariko ubu buryo akenshi butera sensibilité nkeya ya PCR kubera gusohora aside nucleic idahagije.

Kubuza ibikorwa bya polymerase mugihe cya amplification

Muri rusange, kubuzwa gukoreshwa nkigitekerezo cya kontineri kugirango dusobanure ibintu byose biganisha kubisubizo bya PCR. Mu buryo bwa biohimiki rwose, kubuza kugarukira gusa kumikorere ya enzyme, ni ukuvuga, igabanya cyangwa ikabuza guhindura ibicuruzwa biva mu mahanga binyuze mu mikoranire n’urubuga rukora rwa polymerase ya ADN cyangwa cofactor yayo (urugero, Mg2 + kuri Taq ADN polymerase).
Ibigize murugero cyangwa buffers zitandukanye hamwe nibisohoka birimo reagent birashobora guhagarika byimazeyo enzyme cyangwa umutego wa cofactors (urugero EDTA), bityo igakora polymerase nayo iganisha ku kugabanuka cyangwa kubeshya kwa PCR.
Nyamara, imikoranire myinshi hagati yibigize reaction hamwe na acide nucleic acide nayo yagenwe nka 'PCR inhibitor'. Iyo ubunyangamugayo bwakagari buhungabanijwe no kwigunga na aside nucleic irekuwe, imikoranire hagati yicyitegererezo nigisubizo cyacyo hamwe nicyiciro gikomeye kirashobora kubaho. Kurugero, 'scavengers' irashobora guhuza ADN imwe cyangwa ebyiri-ebyiri binyuze mumikoranire idahuje kandi ikabangamira kwigunga no kwezwa mugabanya umubare wintego amaherezo igera mubwato bwa PCR.
Muri rusange, inhibitori za PCR ziboneka mumazi menshi yumubiri hamwe na reagent zikoreshwa mugupimisha kwisuzumisha kwa kliniki (urea mu nkari, hemoglobine na heparin mu maraso), inyongera zimirire (ibinyabuzima, glycogene, ibinure, Ca2 + ion) nibigize ibidukikije (fenolisi) , ibyuma biremereye)

Inhibitori ya PCR yiganje irashobora kuboneka muri bagiteri na selile ya eukaryotic, ADN idafite intego, macromolecules ya ADN ihuza matriche matriche nibikoresho bya laboratoire nka gants na plastike. Isuku rya acide nucleic mugihe cyangwa nyuma yo kuyikuramo nuburyo bwatoranijwe bwo gukuraho PCR inhibitor.
Uyu munsi, ibikoresho bitandukanye byo gukuramo byikora birashobora gusimbuza protocole nyinshi zintoki, ariko 100% kugarura no / cyangwa kweza intego ntabwo byigeze bigerwaho. Inhibitor zishobora kuba zishobora kuba ziri muri acide nucleic yasukuwe cyangwa zishobora kuba zaratangiye gukurikizwa. Ingamba zitandukanye zirahari kugirango zigabanye ingaruka za inhibitor. Guhitamo polymerase ikwiye birashobora kugira ingaruka zikomeye kubikorwa bya inhibitor. Ubundi buryo bwagaragaye bwo kugabanya PCR kubuza ni ukongera imbaraga za polymerase cyangwa gukoresha inyongera nka BSA.
Kubuza ibisubizo bya PCR birashobora kugaragazwa no gukoresha uburyo bwimbere bwo kugenzura ubuziranenge (IPC).
Hagomba kwitonderwa gukuraho reagent zose hamwe nibindi bisubizo mubikoresho byo gukuramo, nka Ethanol, EDTA, CETAB, LiCl, GuSCN, SDS, isopropanol na fenol, muri acide nucleique yitaruye intambwe yo gukaraba neza. Ukurikije ibitekerezo byabo, barashobora gukora cyangwa kubuza PCR.