Ubururu bwa metilene mu gusiga DNA na RNA: uko bukoreshwa muri jeli no ku duhu

Umunyeshuri akoresha jeli ya agarose, akayisiga ubururu bwa metilene, ariko ntabona ikintu aho yari yiteze umurongo ugaragara neza. Mu gice gikurikiyeho cya laboratwari, umushakashatsi warangije impamyabumenyi y'ikirenga asiga agahu ka Northern akoresheje icupa rimwe ry'irangi ry'ubururu, maze mu minota itatu akabona imirongo ya RNA ya ribosome igaragara neza. Umunyeshuri ahita yanzura ko jeli yananiwe. Jeli ntiyananiwe. Ibyo bikorwa byombi byari bitandukanye, kandi irangi rimwe ribisubiza rifite ubushobozi butandukanye bwo gutahura ibipimo bito, ibara ry'inyuma ritandukanye n'amategeko atandukanye agenga ibikurikiraho.

Gusiga DNA n'ubururu bwa metilene no gusiga RNA na bwo bihuje imikorere ya shimi, ariko ntibihuje intego. Muri jeli, irangi rigaragaza ingano ya aside nukileyike iri mu rusobe rufite utwenge. Ku gahu, rigaragaza niba kwimura byagenze neza kandi niba ingano zashyizwe mu nzira zitandukanye zingana. Iyo iryo tandukaniro risobanutse, uburyo bwo kubikora, imipaka yo gutahura ibipimo bito n'imipaka yo guhuza ibikorwa bikurikiraho birumvikana.

Igisubizo kigufi

Ubururu bwa metilene ni irangi rifite imiterere irambuye kandi rifite umuriro w'amashanyarazi mwiza. Ryifatanya na aside nukileyike ridakoze isano ihuriweho ya shimi, kandi uko kwifatanya gushobora gusubizwa inyuma. Ribikora binyuze mu gukururana kw'imiriro y'amashanyarazi na fosifate, kwinjira hagati y'impanga z'ibishingiro no kwifatanya n'umuyoboro muto wa DNA. Kuba uko kwifatanya gushobora gusubizwa inyuma ni byo by'ingenzi. Imirongo ishobora gusigwa, ikarebwa hakoreshejwe umucyo ugaragara, igafotorwa, hanyuma irangi rigakurwamo cyangwa igasukurwa kugira ngo ikoreshwe mu gukora kopi za DNA, guhuza imigozi y'aside nukileyike cyangwa kongera kuyisuzuma hakoreshejwe ikimenyetso.

Ku gusiga DNA y'imigozi ibiri muri jeli ya agarose n'ubururu bwa metilene, uburyo bwihuse busanzwe bukoresha 0.02 percent w/v (200 micrograms kuri mL) mu mazi, gusiga mu gihe cy'iminota hafi 15 no gukuramo irangi mu mazi mu gihe cy'iminota hafi 15, hanyuma jeli ikarebwa ku gasanduku gatanga umucyo wera. Ku duhu, uburyo busanzwe bwa Northern blot bukoresha irangi rya 0.02 kugeza kuri 0.04 percent muri asetate ya sodiyumu hafi ya pH 5.2, gusiga iminota mike nyuma yo kwimura no gufatisha icyitegererezo, hanyuma kwoza n'amazi no gukuramo irangi ryose mbere yo guhuza imigozi. Ubushobozi bwo gutahura ibipimo bito ntiburi hejuru: kuva ku mirongo ya nanograms kugeza ku magana ya nanograms kuri buri murongo wa jeli mu bihe byiza, kandi amabwiriza atanga ibipimo byo kwitondera agaragaza 0.5 kugeza kuri 1 microgram. Iyo ngano iruta cyane cyane ikenewe ku marangi ya bromide ya etidiyumu na SYBR. Ku duhu, nta gipimo kimwe ngenderwaho cyo gutahura molekile imwe ya RNA yandukuwe, kuko irangi ahanini rigaragaza RNA ya ribosome iri ku bwinshi n'ibikoresho byose byashyizwemo. Ntugasimbuze irangi ryagenewe ubwo buryo umuti w'inyunganiramirire ugurishwa mu maduka. Uburyo bwemejwe bufata ko hakoreshwa irangi rifite ubuziranenge bugenewe isesengura, ibisubizo bigumana pH byagenwe, no gukora hatarimo RNase igihe RNA ikoreshwa.

Uko kwifatanya gukora n'aho gushobora kuyobya

Uburyo bw'ingirakamaro bwo kubyumva bugizwe n'ibice bitatu. Icya mbere, urusobe rw'impeta rufite umuriro mwiza rukururwa n'uruti rwa fosifate rufite umuriro mubi. Ubushakashatsi bushingiye ku gupima urumuri bwerekana ko uku gukururana kw'amashanyarazi ari ko kwiganje iyo umunyu ari muke. Naho kwerekana imiterere hifashishijwe mudasobwa no kwiga imigendere ya molekile byerekana ko kwinjira hagati y'impanga z'ibishingiro no kwifatanya n'umuyoboro muto ari uburyo buhamye bwo kwifatanya na DNA y'imigozi ibiri, nk'uko byegeranyijwe mu bushakashatsi bushingiye ku gupima urumuri ku kwifatanya k'ubururu bwa metilene na DNA. Icya kabiri, hari aho GC ikundwa kurusha ibindi: kwerekana imiterere hifashishijwe mudasobwa bishyigikira ibice bya CpG na GpC, kandi igerageza rigaragaza ko kwifatanya bikunze kuba ku migozi ibiri ikungahaye kuri GC, mu bushakashatsi ku byo ubururu bwa metilene bukunda kwinjiramo hagati y'ibishingiro. Ariko n'imigozi ibiri ya AT iraryifatanyaho. Gusiga bisanzwe ntibihitamo uruhererekane runaka rw'ibishingiro. Icya gatatu, DNA y'umugozi umwe na RNA byifatanya cyane cyane binyuze mu gukururana kw'amashanyarazi no mu gutondekana hejuru y'ibishingiro bigerwaho n'ibice by'imigozi ibiri. Ni yo mpamvu ubukana bw'ibara buhinduka bitewe n'uburebure, uruhererekane rw'ibishingiro n'imiterere ya kabiri.

Ibyo bigira ingaruka ebyiri. Kubera ko kwifatanya kudakorwa n'isano ihuriweho ya shimi, irangi riragenda iyo ryogejwe. Imirongo yo muri polyacrylamide ihita icika ibara keretse ifatishijwe ku bushake. Hari uburyo bwihariye bwo gufatisha irangi burundu, bwashyizweho kubera ko gusiga bisanzwe bimara igihe gito, nk'uko Soto na Draper babisobanuye kuri jeli za PAGE. Kubera ko irangi rituma ibikorwa bya shimi biterwa n'umucyo bishoboka, kumurikira DNA yasizwe igihe kirekire n'umucyo mwinshi ahari ogisijeni bishobora gutuma guanine ihinduka binyuze mu kwamburwa electron, bikagera n'aho bica igice cy'umugozi wa DNA. Iyo mikorere isobanuwe mu bushakashatsi ku ihinduka rya guanine riterwa n'umucyo hifashishijwe ubururu bwa metilene. Kureba mu gihe gito hakoreshejwe umucyo wera birinda kwangirika kuzwi guterwa na UV mu buryo busanzwe bukoresha bromide ya etidiyumu, kandi iyo nyungu ni nyakuri iyo hagamijwe kugarura DNA ngo ikoreshwe. Umucyo ugaragara wangiza gake, ariko si ukuvuga ko nta ngaruka ugira. Gabanya igihe cyo kumurika igihe DNA izakoreshwa mu gukora kopi. Ku bisobanuro birambuye bya shimi iterwa n'umucyo bishyigikira uko kwitondera, reba inzira ebyiri za shimi iterwa n'umucyo mu bururu bwa metilene. Ku miterere ibiri ijyanye no gutanga cyangwa kwakira electron n'imiterere y'umuriro w'amashanyarazi bisobanura impamvu imiterere yambuwe electron yifatanya, naho iyakiriye electron ntibyifatanye, reba shimi y'imiterere n'ibiranga ubururu bwa metilene.

Igikorwa cya mbere: gusiga imbere muri jeli

Gusiga jeli bisubiza ikibazo kimwe: ese muri uyu murongo harimo aside nukileyike ihagije ngo ibonwe n'amaso? DNA y'imigozi ibiri muri agarose ni yo yizwe neza kurusha izindi. Uburyo bwa mbere bwa Flores na bagenzi be bwo gusiga jeli n'ubururu bwa metilene bwemeje uburyo bwihuse bwa 0.02 percent kuri agarose ya 1 percent, kandi bwerekana ko DNA yashoboraga kugarurwa no gukorerwa kopi nyuma yaho. Amabwiriza amwe y'uwabutanze agaragara ku rupapuro rwa IBI Scientific rw'igicuruzwa cy'ubururu bwa metilene, rugaragaza irangi rya 0.04 percent rikoreshwa ku duhu n'uburyo bwihuse bwo gusiga jeli iruhande rumwe rw'urundi.

Uko bikorwa:

1. Koresha jeli nk'uko bisanzwe. Yisige nyuma yo gutandukanya ibikoresho hakoreshejwe amashanyarazi, atari mu gihe bikorwa, keretse kwimura cyangwa gutandukanya neza byemejwe ku buryo bwahinduwe.
2. Yibize mu bururu bwa metilene bwa 0.02 percent w/v buri mu mazi yatunganijwe hakoreshejwe guhumeka no gukonjesha umwuka, uyinyeganyeze buhoro mu gihe cy'iminota hafi 15.
3. Kuramo irangi uhindura amazi inshuro nyinshi, mu gihe cy'iminota hafi 15 yose hamwe, kugeza ibara ry'inyuma ry'urusobe rigabanutse maze imirongo ikagaragara neza.
4. Yifotore vuba iri ku gikoresho gitanga umucyo wera.

Agarose na polyacrylamide byitwara mu buryo butandukanye. Agarose igumana irangi mu rusobe, bityo gukuramo irangi ni cyo gikorwa gitinda. Jeli ntoya z'umubyimba za polyacrylamide zisigwa vuba, ariko na zo zitakaza irangi vuba. Ibyo bifasha gukata igice kigamije gukoreshwa, ariko bikabangamira kubika inyandiko z'ibisubizo. Oligonukileyotide zakozwe muri laboratwari ziri muri polyacrylamide zishobora kugaragazwa n'irangi rimwe rya 0.02 percent mu minota 20 kugeza kuri 30, hakoreshejwe kwoza kenshi n'amazi, hakurikijwe uburyo bwa PAGE busanzwe bwo mu bwoko bwa Current Protocols. Hari ubushakashatsi bwatangaje uburyo bukoresha irangi ryoroheje cyane hafi ya 0.0002 percent muri TBE yoroshywe, mu gutegura oligonukileyotide mu gihe cy'amasaha menshi, ariko ibimenyetso bibushyigikira ni bike.

Itandukaniro hagati ya DNA na RNA ni ingenzi. DNA y'imigozi ibiri yifatanya cyane kandi mu buryo bufite imiterere isobanutse. RNA na DNA y'umugozi umwe na byo birasigwa, ariko bikagira ibara ryoroshye kandi ridashobora guteganywa neza. Kuri jeli za RNA za agarose zirimo formaldehyde, laboratwari nyinshi ntizisiga irangi ry'ubururu muri jeli; zibanza kwimurira RNA ku gahu, zikayisigira aho. Ubwo buryo busimbuza ikimenyetso kidakomeye cyo muri jeli ikimenyetso kigaragara neza ku gahu, bugatanga n'inyandiko y'ingano zashyizwemo.

Igikorwa cya kabiri: gusiga ku duhu

Gusiga ku gahu bisubiza ikindi kibazo: ese kwimura byagenze neza, kandi inzira zitandukanye ziragereranywa? Uburyo busanzwe ni ubwa Herrin na Schmidt bwo gusiga Northern blot hakoreshejwe irangi rishobora gukurwamo. Mu ntangiriro bwakoreshaga irangi rya 0.04 percent muri asetate ya sodiyumu ya 50 mM kuri pH 5.0 mu minota 2 kugeza kuri 3, hagakurikiraho kwoza n'amazi. Irangi ryashoboraga gukurwamo na SDS yoroshywe muri SSPE mbere yo guhuza imigozi.

Uburyo bugezweho bukoreshwa cyane bukorwa gutya:

1. Imurira RNA cyangwa DNA yatandukanyijwe imigozi kuri nylon cyangwa nitrocellulose, hanyuma ubifatishishe UV cyangwa ubushyuhe kugira ngo icyitegererezo kidatakara mu gihe cyo gusiga.
2. Si ngombwa ariko birasanzwe: tosa agahu muri aside asetike ya 5 percent mu minota hafi 15.
3. Siga iminota 5 kugeza kuri 10 ukoresheje ubururu bwa metilene bwa 0.02 kugeza kuri 0.04 percent muri asetate ya sodiyumu ya 0.3 kugeza kuri 0.5 M hafi ya pH 5.2.
4. Oza kandi ukuremo irangi mu mazi mu minota 5 kugeza kuri 15, fata inyandiko y'ibisubizo, hanyuma ukuremo irangi risigaye mbere yo guhuza imigozi.

Nylon ifite umuriro w'amashanyarazi mwiza ni yo ikoreshwa cyane muri Northern. Nitrocellulose na yo ikorana n'ubwo buryo bukoresha aside asetike na asetate. Ishusho ikoreshwa mu kwemeza ko kwimura byagenze neza, kugereranya imirongo ya 28S na 18S (cyangwa 23S na 16S kuri RNA ya bagiteri), gushyira ibimenyetso ku bipimo ngenderwaho by'uburebure no ku bindi bipimo ngenderwaho, no kubika inyandiko igereranya RNA yose yashyizwemo. Uburyo bwa Northern bwatangajwe mu bushakashatsi bupima imirongo ya RNA ya ribosome yasizwe kugira ngo bugenzure ingano zashyizwemo, harimo n'ubushakashatsi mu by'amata bwakoresheje imirongo ya RNA ya ribosome yasizwe ubururu bwa metilene mu kugereranya ingano. Uduhu twa Southern dushobora kugaragazwa muri ubwo buryo, ariko irangi ryamenyekanye cyane mu kugenzura ingano zashyizwemo no kwimura muri Northern. DNA ya Southern itandukanywa imigozi mbere yo kwimurwa, bityo imibare y'ubushobozi bwo gutahura ibipimo bito muri jeli ntishobora gukoreshwa uko yakabaye.

Imipaka yo gutahura ibipimo bito n'ibara ry'inyuma: aho ubururu budakora neza

Igereranya rikurikira ritanga ibipimo bigereranyije ku bushake. Imikorere y'irangi iterwa n'ijanisha rya jeli, ubugari bw'umurongo, ibikoresho by'urumuri n'ubwitonzi mu gukuramo irangi. Fata buri mubare nk'urwego rw'ingano, aho kuwufata nk'igipimo ntarengwa cyemejwe.

Kugenzura ibara ry'inyuma ni byo bigena niba ikimenyetso kidakomeye gishobora gukoreshwa. Irangi ritifatanyije rigira ibara rikomeye cyane; bityo gutandukanya imirongo n'inyuma bituruka ku gukuramo irangi ritifatanyije utakuyeho iryifatanyije. Kongera ubwinshi bw'irangi ntibikunze kongera ubushobozi bwo gutahura ibipimo bito. Ahanini byongera igihe cyo kurikuramo. Gerageza uruhererekane rw'ibipimo bigenda byoroha ukoresheje jeli, agahu na kamera bizakoreshwa nyirizina, kandi ukoreshe gusa urwego aho ubukana bwose bw'ishusho yapimwe bukomeza kugendana n'ingano yashyizwemo. Ubururu bwa metilene bufasha kugereranya ingano zashyizwemo mu buryo butanga ibipimo bigereranyije. Si uburyo bwo gupima neza mu ntera nini y'ingano, kuko ubushobozi bw'agahu, imikorere yo kwimura, kuzura no gukuramo irangi bitandukana hagati y'inzira.

Imipaka yo guhuza ibikorwa: ibishobora gukorwa nyuma yo gusiga

Gukora kopi za DNA nyuma yo gusiga ubururu byaragaragajwe. Flores na bagenzi be bakase imirongo ya agarose yasizwe, bagarura DNA, barayifatanya maze bayinjiza mu turemangingo. Batangaje ko gukora kopi byagenze neza kurusha nyuma yo gukoresha bromide ya etidiyumu na UV. Impamvu ishoboka ni ukwangirika guke guterwa n'umucyo, atari inyungu ituruka ku irangi ubwaryo. Gabanya igihe DNA imara mu mucyo wera, kandi usukure DNA yagaruwe ukoresheje uburyo busanzwe bwo kuyikura muri jeli.

Ibimenyetso byerekeye PCR no gusoma uruhererekane rw'ibishingiro ni bike. Irangi risigaye ryifatanya na aside nukileyike; bityo rikuremo kandi usukure mbere y'ibikorwa bikoresha anzime. Biteganyijwe ko DNA yasukuwe neza ivuye muri jeli yasizwe ubururu yakora, ariko ingano y'irangi risigaye ishobora kwihanganirwa na polimeraze zitandukanye n'uburyo bwa shimi bwo gusoma uruhererekane ntipimwe neza. Ntukabifate nk'ibyemejwe.

Guhuza imigozi kuri blot birashoboka iyo uruhererekane rwubahirijwe neza: kwimura, gufatisha, gusiga, gufata inyandiko y'ibisubizo, gukuramo irangi, guhuza imigozi. Urwo ni rwo ruhererekane rwateguwe na Herrin na Schmidt. Gusiga mbere yo kwimura cyangwa kudakuramo irangi ni ubundi buryo kandi bugomba kwemezwa ukwabwo. Nyuma yo gukuramo irangi, gukuramo ibimenyetso bisanzwe no kongera gusuzuma hakoreshejwe ibindi bimenyetso bikomeza nk'uko bisanzwe. Ku gusiga ku rwego rw'uturemangingo hakoreshejwe amabara yo muri uwo muryango, n'impamvu uburyo bukoreshwa ku gice cyafatishijwe budashobora kwimurirwa uko bwakabaye ku turemangingo tuzima, reba igitabo cy'amashusho y'uburyo bwo gusiga mu kureba na mikorosikopi.

Gukata umurongo birakora kuri agarose igamije kugarura ibikoresho no kuri oligonukileyotide zo muri PAGE zagaragajwe n'ubururu, igihe uburyo bwo gufatisha burundu bwirinzwe. Gukura ibikoresho muri jeli hakoreshejwe amashanyarazi nyuma yo gukuramo irangi birashoboka mu rwego rwa shimi ku irangi ryifatanya mu buryo bushobora gusubizwa inyuma, ariko amakuru y'igerageza rigenzuwe ku ngano igarurwa hifashishijwe ubururu bwa metilene ni make. Nyuma yo gukura ibikoresho muri jeli, bikuremo umunyu cyangwa ubikure mu gisubizo binyuze mu kubituma biba ibikomeye.

Ibibazo bikunda kongera kubaho

Imirongo ya jeli igaragara gake akenshi isobanura ko ingano ari nto cyane ku irangi rigaragazwa n'ibara, cyane cyane kuri DNA ngufi y'umugozi umwe cyangwa RNA. Shyiramo ingano nyinshi, ukoreshe irangi rishya, wongere igihe cyo gusiga mbere yo kongera ubwinshi bw'irangi, kandi uhagarike gukuramo irangi hakiri kare. Jeli yose ifite ibara ry'ubururu ridatuma imirongo itandukana n'inyuma akenshi isobanura gusiga birenze urugero cyangwa kudahindura amazi bihagije. Kuba imirongo ya PAGE icika ibara ni imikorere ya shimi yitezwe, si uko irangi ryananiwe. Fata amafoto vuba. Ibara ry'inyuma ritaringaniye ku gahu rishobora guterwa n'imizingo, utubyimba tw'umwuka, ibimenyetso by'intoki, kunyeganyeza kutaringaniye cyangwa kwimura kutabaye hose kimwe. Ntukabisobanure nk'ingano zashyizwemo zitangana udafite igerageza rigenzura rishingiye ku guhuza imigozi. Imirongo ya RNA ya ribosome itagaragara neza kandi isa n'iyasakaye ishobora gusobanura ko RNA yangiritse, ariko ishobora no guterwa no kwimura kutarangiye, RNA yose hamwe ari nke cyane (inzira zisanzwe zishyirwamo kuva kuri micrograms nke kugeza ku mirongo ya micrograms), cyangwa gukuramo irangi birenze urugero. Irangi rigaragaza RNA ya ribosome iri ku bwinshi. Ntirigaragaza molekile yihariye ya RNA yandukuwe. Icyo ni umurimo w'ikimenyetso gikoreshwa mu kuyishakisha.

Umutekano no gukoresha utarengeje ibyo ibimenyetso byemeza

Ubururu bwa metilene bworoshye gukoresha kurusha bromide ya etidiyumu, ariko si ikintu kidafite ingaruka. Urupapuro rw'amakuru y'umutekano rugezweho rwa MilliporeSigma rushyira irangi mu Cyiciro cya 4 cy'uburozi bwihuse iyo rinyujijwe mu kanwa, rukavuga ko kwangiza biramutse rimizwe, kandi ko imyanda yaryo igomba kunyuzwa mu buryo bwemewe, nk'uko biri mu rupapuro rwa Sigma-Aldrich rw'amakuru y'umutekano y'ubururu bwa metilene. Bromide ya etidiyumu ifite imiburo ikomeye kurushaho ku byerekeye kwangiza ibikoresho by'irondakoko, harimo n'ingaruka zikekwa ku turemangingo tw'imyororokere zivugwa mu rupapuro rwa Sigma-Aldrich rw'amakuru y'umutekano ya bromide ya etidiyumu. Jeli n'ibisubizo biyirimo bikunze gutandukanywa n'indi myanda nk'imyanda ya shimi iteje akaga, nk'uko bivugwa mu gitabo cya Thermo Fisher cy'amabwiriza y'amabara asiga aside nukileyike. Kurikiza urupapuro rw'amakuru y'umutekano n'amategeko y'ikigo kuri byombi. Bika irangi ryumye rifunze neza kandi ritose, wongere ukoreshe irangi ryateguwe gusa uko amabwiriza abyemera, ubike uduhu twasizwe twumye hagati y'impapuro z'iyungurura, kandi ufotore jeli mbere y'uko imirongo ikwirakwira ikabura.

Ibuka interuro ngenderwaho ku meza ya laboratwari. Jeli zisuzuma niba ingano nyinshi zigaragara mu mucyo wera. Uduhu dusuzuma kwimura n'ingano zashyizwemo mbere y'uko ikimenyetso gikora umurimo wo gutahura ibipimo bito. Irangi rimwe rikora byombi, rifite ubushobozi butari hejuru bwo gutahura ibipimo bito, kwifatanya gushobora gusubizwa inyuma n'uruhererekane rw'ibikorwa rugomba kubahirizwa neza.