Ni bangahe Inyandikorugero Twakagombye Kongera kuri PCR Yanjye?

Nubwo mubitekerezo, molekile imwe yicyitegererezo yaba ihagije, umubare munini cyane wa ADN ikoreshwa muri PCR isanzwe, urugero, kugeza kuri 1 µg ya ADN y’inyamabere y’inyamabere na pg 1 za ADN ya plasmide. Umubare mwiza uterwa ahanini numubare wa kopi yintego ikurikiranye, kimwe nuburyo bugoye.

Niba inyandikorugero ntoya cyane ikoreshwa, kwiyongera gukwiranye numubare wa amplification cycle bizakenerwa kugirango ubone ibicuruzwa bihagije. Taq polymerase ikoreshwa mubigeragezo byinshi bya PCR ntabwo igaragaramo imikorere yo gukosora (3′-5 ′ ibikorwa bya exonuclease); bityo, amakosa abaho mugihe cya amplification ntashobora gukosorwa. Umubare munini wizunguruka, niko byiyongera cyane kwongera ibicuruzwa bitagira inenge bizaba. Niba, kurundi ruhande, ingano yicyitegererezo ni ndende cyane, amahirwe ya primers yegeranye nizindi (ntabwo ijana ku ijana zishimwe) zikurikirana, kimwe no gushiraho primer dimers, biziyongera, bizavamo kwiyongera kwa Ibicuruzwa. Mubihe byinshi, ADN itandukanijwe numuco wimikorere cyangwa mikorobe hanyuma igakoreshwa nkicyitegererezo cya PCR. Nyuma yo kwezwa, birakenewe kumenya ubunini bwa ADN kugirango ubashe gusobanura ingano isabwa kugirango PCR ishyirwe. Mugihe agarose gel electrophorei irashobora gutanga ikigereranyo, ubu buryo ntiburi kure. UV-Vis spectrophotometrie yashyizweho nkurwego rwa zahabu rwo kugereranya acide nucleic; ubu buryo butaziguye kandi bworoshye kandi bwihuse bupima kwinjiza icyitegererezo kuri 260 nm, kandi kwibanda kubarwa hifashishijwe ibintu bihinduka.

Niba intumbero ya ADN iri hasi cyane, ariko (<1 µg / mL dsDNA), cyangwa niba yaranduye ibintu na byo byinjira mu ntera ya 260 nm (urugero RNA, proteyine, umunyu), ubu buryo buzagera aho bugarukira. Mugihe cyibitekerezo bike cyane, ibyasomwe bizahita bidahwitse kuburyo bidashobora gukoreshwa, kandi kwanduza bizatuma (rimwe na rimwe binini) birenze urugero agaciro nyako. Muri iki kibazo, umubare ukoresheje fluorescence urashobora kwerekana ubundi buryo. Ubu buhanga bushingiye ku gukoresha irangi rya fluorescent ihuza cyane na dsDNA gusa uruganda rugizwe na acide nucleic aside hamwe n irangi rishimishwa numucyo, hanyuma bikazasohora urumuri rwuburebure buke buke. Hano, ubukana bwikimenyetso cya fluorescente buringaniye nubunini bwa ADN, kandi kugirango hamenyekane intumbero isuzumwa bijyanye numurongo usanzwe. Ibyiza byubu buryo bishingiye ku mwihariko w’ubucuti, ukuyemo ingaruka zituruka hanze ziterwa no kwanduzwa, ndetse nubushobozi buvuyemo bwo kumenya ADN nkeya cyane. Uburyo bukwiye bwuburyo bumwe buterwa ahanini nicyitegererezo hamwe nubuziranenge; mubihe byinshi birashobora no kuba byiza gukoresha uburyo bwombi muburyo bubangikanye.