Kunyangwe mudzidziso, morekuru rimwe retemplate raizokwana, huwandu hwakakura kwazvo hweDNA hunowanzo shandiswa kuPCR yekare, semuenzaniso, kusvika pa1 µg yeDNA yemammalian genomic uye zvishoma se1 pg yeplasmid DNA. Iyo yakakwana huwandu hunoenderana zvakanyanya nenhamba yemakopi ekutevedzana kwechinangwa, pamwe nekuoma kwayo.
Kana template diki diki ikashandiswa, kuwedzera kunoenderana nenhamba yeamplification cycles ichadikanwa kuti uwane huwandu hwakakwana hwechigadzirwa. A Taq polymerase iyo inoshandiswa kune yakawanda PCR miedzo haina basa rekugadzirisa (3'-5′ exonuclease basa); saka, kukanganisa kunoitika panguva yekuwedzera hakugone kugadziriswa. Iyo yakakwira huwandu hwema cycles, kunowedzera kuwanda kwechigadzirwa chisina kukwana kuchave. Kana, kune rumwe rutivi, huwandu hwe template hwakanyanya, mukana we primers annealing kune mamwe (kwete zana kubva muzana complimentary) sequences, pamwe nekuumbwa kwe primer dimers, ichawedzera, izvo zvinozoguma nekuwedzera kwe. ne-zvigadzirwa. Muzviitiko zvakawanda, DNA inoparadzaniswa nesero tsika kana kubva kune tupukanana uye inozoshandiswa sePCR template. Kutevera kucheneswa, zvinodikanwa kuona kusangana kweDNA kuti ikwanise kutsanangura huwandu hunodiwa pakuseta PCR. Kunyange zvazvo agarose gel electrophoresis inogona kushanda kupa fungidziro, nzira iyi iri kure nekururama. UV-Vis spectrophotometry yakamiswa seye goridhe mwero weiyo quantification ye nucleic acids; iyi yakananga uye naizvozvo iri nyore uye inokurumidza nzira inoyera kunyura kwemuenzaniso pa 260 nm, uye kuisa pfungwa kunoverengwa nerubatsiro rwekushandura chinhu.
Kana iyo DNA concentration yakaderera zvikuru, zvisinei (<1 µg/mL dsDNA), kana kana yakasvibiswa nezvinhu zvinotorawo mu260 nm range (eg RNA, protein, salts), iyi nzira ichasvika painogumira. Panyaya yekudzika kwakadzika kwazvo, kuverenga kuchakurumidza kuve kusina kurongeka kuti kuve kwekushandisa, uye kusvibiswa kunotungamira kune (dzimwe nguva yakakura) kuwedzeredza kukosha chaiko. Muchiitiko ichi, quantification uchishandisa fluorescence inogona kupa imwe nzira. Iyi nzira inobva pakushandiswa kwedhayi yefluorescent inosunga zvakananga ku dsDNA chete yakaoma inosanganisira nucleic acid uye dhayi inonakidzwa nechiedza, uye inozobudisa chiedza chepamusoro zvishoma. Pano, kusimba kwechiratidzo chefluorescent kunoenderana nehuwandu hweDNA, uye yekuona iyo yakasungwa inoongororwa maererano neyakajairwa curve. Zvakanakira nzira iyi zvinotsamira pane chaiyo yechisungo, iyo isingabatanidzi maitiro ekunze akaunzwa nekusvibiswa, pamwe nekukwanisa kunoguma kwekuona kuderera kwakanyanya kweDNA. Kukodzera kweimwe nzira kunoenderana zvakanyanya nemuenzaniso wekutarisa uye kuchena; muzviitiko zvakawanda zvingatova zvakanaka kushandisa nzira dzose dziri mbiri dzakafanana.
Nguva yekutumira: Nov-30-2022