Nangona kwithiyori, imolekyuli enye yetemplate inokwanela, izixa ezikhulu kakhulu ze-DNA ziqhele ukusetyenziswa kwi-PCR yakudala, umzekelo, ukuya kuthi ga kwi-1 µg ye-genomic mammalian DNA kunye nencinci njenge-1 pg ye-plasmid DNA. Elona xabiso lifanelekileyo lixhomekeke kakhulu kwinani leekopi zolandelelwano ekujoliswe kulo, kunye nokuntsonkotha kwayo.
Ukuba ithemplate encinci isetyenzisiweyo, ukunyuka okuhambelanayo kwinani lemijikelezo yokukhulisa kuya kufuneka ukufumana isixa esaneleyo semveliso. I-Taq polymerase esetyenziswa kwiimvavanyo ezininzi ze-PCR ayinawo umsebenzi wokulungisa (3'-5′ umsebenzi we-exonuclease); ngoko ke, iimpazamo ezenzeka ngexesha lokukhulisa azinako ukulungiswa. Okukhona liphezulu inani lemijikelo, kokukhona kuxhaphake ngakumbi ukukhulisa imveliso ephosakeleyo. Ukuba, kwelinye icala, inani le template liphezulu kakhulu, amathuba okuba iiprimers annealing kwezinye (hayi ikhulu lepesenti elincomekayo) ulandelelwano, kunye nokwakhiwa kwe-primer dimers, kuya kwanda, okuya kubangela ukukhulisa ngemveliso. Kwiimeko ezininzi, i-DNA yodwa kwiinkcubeko zeeseli okanye kwii-microorganisms kwaye emva koko isetyenziswe njengetemplate ye-PCR. Ukulandela ukuhlanjululwa, kuyimfuneko ukumisela ukugxininiswa kwe-DNA ukuze ukwazi ukuchaza umthamo ofunekayo ukuseta i-PCR. Ngelixa i-agarose gel electrophoresis inokusebenza ukunika uqikelelo, le ndlela ayichanekanga. I-UV-Vis spectrophotometry iye yasekwa njengomgangatho wegolide wokulinganisa i-nucleic acids; le ndlela ngokuthe ngqo kwaye ngoko ke ilula kwaye ikhawuleza indlela yokuthatha isampuli kwi-260 nm, kwaye ugxininiso lubalwa ngoncedo lokuguqulwa kwezinto.
Ukuba i-DNA concentration iphantsi kakhulu, nangona kunjalo (< 1 µg/mL dsDNA), okanye ukuba ingcolisekile ngezinto ezifunxa kwakhona kuluhlu lwe-260 nm (umz. RNA, protein, salts), le ndlela iya kufikelela kwimida yayo. Kwimeko yogxininiso oluphantsi kakhulu, ufundo kungekudala luya kuba lungachanekanga kakhulu ukuba lungasetyenziswa, kwaye ungcoliseko luya kukhokelela (ngamanye amaxesha lukhulu) uqikelelo olugqithisileyo lwelona xabiso lokwenene. Kule meko, ubungakanani be-fluorescence bunokubonisa enye indlela. Obu buchwephesha busekwe kusetyenziso lwedayi ye-fluorescent ebophelela ngokukodwa kwi-dsDNA kuphela entsonkothileyo equka i-nucleic acid kunye nedayi echulumancisayo kukukhanya, kwaye iya kuthi emva koko ikhuphe ukukhanya kobude obungaphezulu kancinci. Apha, ubukhulu besignali ye-fluorescent ihambelana nomlinganiselo we-DNA, kwaye ukugqiba ugxininiso luvandlakanywa ngokubhekiselele kwi-curve eqhelekileyo. Iingenelo zale ndlela zixhomekeke kwi-speciality ye-bond, engabandakanyi iimpembelelo zangaphandle ezivezwa kungcoliseko, kunye nesiphumo sokukwazi ukubona ukugxila okuphantsi kakhulu kwe-DNA. Ukufaneleka kwayo nayiphi na indlela kuxhomekeke ikakhulu kugxininiso lwesampulu kunye nobunyulu; kwiimeko ezininzi kunokude kucebiseke ukusebenzisa iindlela zombini ngokuhambelana.
Ixesha lokuposa: Nov-30-2022