Kodi Tiziwonjeza Zambiri Bwanji pa PCR Reaction yanga?

Ngakhale m'malingaliro, molekyu imodzi ya template ingakhale yokwanira, kuchuluka kwakukulu kwa DNA kumagwiritsidwa ntchito ngati PCR yachikale, mwachitsanzo, mpaka 1 µg ya DNA ya mammalian genomic komanso 1 pg ya plasmid DNA. Kuchuluka koyenera kumadalira makamaka kuchuluka kwa makope a mndandanda wazomwe mukufuna, komanso zovuta zake.

Ngati template yaying'ono ikugwiritsidwa ntchito, kuwonjezereka kofanana kwa kuchuluka kwa mizere yokulitsa kudzafunika kuti mupeze kuchuluka kokwanira kwazinthu. Taq polymerase yomwe imagwiritsidwa ntchito poyesa zambiri za PCR ilibe ntchito yokonza (3'-5′ exonuclease ntchito); motero, zolakwika zomwe zimachitika pakukulitsa sizingakonzedwe. Kuchulukirachulukira kwa mizere, m'pamenenso kuchulukitsidwa kwa zinthu zolakwika kumachulukirachulukira. Ngati, kumbali ina, kuchuluka kwa template ndikwambiri, kuthekera kwa zoyambira zoyambira kutsatizana ndi zina (osati zana limodzi mwazovomerezeka) zotsatizana, komanso kupanga ma dimers oyambira, kudzawonjezeka, zomwe zimabweretsa kukulitsa kwa zopangira. Nthawi zambiri, DNA imasiyanitsidwa ndi zikhalidwe zama cell kapena ku tizilombo tating'onoting'ono ndipo kenako imagwiritsidwa ntchito ngati template ya PCR. Kutsatira kuyeretsedwa, ndikofunikira kudziwa kuchuluka kwa DNA kuti athe kufotokozera voliyumu yomwe ikufunika pakukhazikitsa kwa PCR. Ngakhale kuti agarose gel electrophoresis ikhoza kupereka chiŵerengero, njira iyi si yolondola. UV-Vis spectrophotometry yakhazikitsidwa ngati muyeso wagolide wowerengera ma nucleic acid; Njira iyi yachindunji komanso yosavuta komanso yofulumira imayesa kuyamwa kwachitsanzo pa 260 nm, ndipo ndende imawerengedwa mothandizidwa ndi chinthu chotembenuka.

Ngati chiwerengero cha DNA chili chochepa kwambiri, komabe (< 1 µg/mL dsDNA), kapena ngati chakhudzidwa ndi zinthu zomwe zimayamwanso mu 260 nm range (monga RNA, mapuloteni, mchere), njirayi idzafika malire ake. Pankhani yotsika kwambiri, zowerengera posachedwapa zidzakhala zosalondola kwambiri kuti zigwiritsidwe ntchito, ndipo kuipitsidwa kudzatsogolera ku (nthawi zina kwakukulu) kuyerekezera mtengo weniweni. Pankhaniyi, quantification pogwiritsa ntchito fluorescence angapereke njira ina. Njirayi imachokera pakugwiritsa ntchito utoto wa fulorosenti womwe umamangiriza makamaka ku dsDNA kokha zovuta zomwe zimakhala ndi nucleic acid ndi utoto zimasangalatsidwa ndi kuwala, ndipo pambuyo pake zidzatulutsa kuwala kwa utali wokwera pang'ono. Apa, mphamvu ya siginecha ya fulorosenti ndi yofanana ndi kuchuluka kwa DNA, ndipo kuti mudziwe kuchuluka kwake kumawunikidwa mogwirizana ndi mapindikidwe wamba. Ubwino wa njirayi umakhala pa kutsimikizika kwa chomangiracho, chomwe sichiphatikiza zikoka zakunja zomwe zimayambitsidwa ndi kuipitsidwa, komanso kuthekera kodziwira kutsika kwambiri kwa DNA. Kuyenerera kwa njira iliyonse kumadalira makamaka chitsanzo cha ndende ndi chiyero; nthawi zambiri zingakhale bwino kugwiritsa ntchito njira zonse ziwiri zofanana.