Chii icho PCR Chingaita NeDNA Sequencing uye Kuwedzera Genesesi
Inonzi polymerase chain reaction ( PCR ) inonzi molecular genetic method yekuita makopi akawanda ejeni uye inowanikwawo muitiro wekugadzirisa maitiro.
Ko Polymerase Chein Reaction Inoshanda Sei?
Gene makopi anoshandiswa achishandisa shanduro yeDNA, uye teknolojia yakakosha zvokuti inoita makopi akawanda kubva kune imwe chete yejeni inowanika muenzaniso. PCR kuwedzerwa kwegene kuita mamiriyoni emakopi, kunobvumira kuonekwa nekuzivikanwa kwekugadzirisa kwemajini kushandisa nzira dzinoonekwa zvichienderana nekukura uye kutengesa (+ kana -) kwechikamu cheDNA.
Pasi pemamiriro ezvinhu akachengetedzwa, zvikamu zviduku zveDNA zvinoumbwa nemakemikari anozivikanwa seDNA polymerases, ayo anowedzera zvinonzi deoxynucleotides (dNTPs) kune chimwe chikamu cheDNA chinonzi "template." Kunyange zvidimbu zviduku zveDNA, zvinonzi "primers" zvinoshandiswa sekutanga kwe polymerase. Zvipfeko zvikamu zviduku zvakagadzirwa nemunhu zveDNA (oligomers), kazhinji pakati pe15 ne 30 nucleotide kwenguva refu. Zvinoshandiswa nokuziva kana kufungidzira kupfupika kweDNA kuenzaniswa pamigumo yegene iri kukura. Munguva yePCR, DNA iri kuvharidzirwa inopisa uye mapfundo maviri akaparadzana. Painotonhora, inotanga kusungira ku template (inonzi annealing) uye inoita nzvimbo yekuti polymerase itange.
Iyo PCR Technique
Iyo polymerase chain reaction (PCR) yakagoneka kuburikidza nokuwanikwa kwe thermophiles uye thermophilic polymerase enzyme (enzyme dzinoramba dzichigadzikana nekugadziriswa mushure mekunge ichipisa pamatambo makuru).
Matanho anobatanidzwa mumhando yePCR ndeyotevera:
- Musanganiswa unogadzirwa, uye yakanyatsogadziriswa kweDNA template, polymerase enzyme, primers, uye dNTPs. Kukwanisa kupisa musanganiswa pasina kutyisidzira enzyme kunobvumira kudengesa kwekaviri helix yeDNA sampu pamatsetse emvura inenge 94 degrees Celsius.
- Kutevera kuratidzira, samuenzaniso yakanatswa kusvika kune imwe nzvimbo yakaenzana, inenge ma-54 degrees, iyo inofambisa kuvhara (kusunga) kwekutanga kune DNA imwe chete yakanyanyiswa.
- Muchikamu chechitatu chekufamba, sampu inodzokorora zvakare kusvika kune madigiri 72, iyo inotonhorera yeTaq DNA Polymerase, nokuda kwekuwedzera. Pakati pezvinyorwa, DNA polymerase inoshandisa chigadziro chekutanga cheDNA se template kuwedzera dNTPs kuwedzera kumagumo matatu ekutanga uye kugadzira chikamu cheDNA mbiri-yakagadzirirwa munharaunda yezvibereko zvekufarira.
- Zvipfeko zvakave zvakanyatsoenderana neDNA zvakaenzanirana izvo zvisina kufanana zvakafanana hazvisati zvichinyorwa pamadhora 72, nokudaro zvichidzivirira kukwana kusvika kune jeni rekufarira.
Iyi nzira yekuratidza, kushandiswa uye kuburitsa kunodzokorora kazhinji (30-40) nguva, zvichidaro zvichiwedzera kujekesa nhamba yemakopi emagetsi anodiwa mune musanganiswa. Kunyange zvazvo izvi zvingave zvakanyanyisa kana zvichiitwa nemaoko, sampu inogona kugadzirirwa uye inobatanidzwa muThermocycler inogadziridzwa, ikozvino inowanikwa mune dzakawanda maserocular laboratories, uye kukwanisa kwePCR kushandiswa kunogona kuitwa mumaawa 3-4.
Nhanho imwe neimwe yekudzivisa inogumisa nzira yehutungamiri hwehutambo hwakapfuura, nokudaro ichichera chigamba chitsva cheDNA uye ichichichengetedza kusvika kuhukuru hwemajini anoda.
Nguva yenguva yakatarisa inogona kuitwa kwenguva yakareba kana shoma zvichienderana nekukura kwegene rekufarira, asi pakupedzisira, kuburikidza nekudzokorora kwepurogiramu yePCR, huwandu hwema templates huchasongedzwa kuhukuru hwegene rekufarira chete, sezvo richave rakagadzirwa kubva mumiti yezvinhu zvose zviri zviviri.
Kune zvinhu zvakasiyana-siyana zvakasiyana zvePCR inobudirira iyo inogona kushandiswa kuwedzera migumisiro. Inonyanya kushandiswa nzira yekuedza kuvepo kwePC product ndeye agarose gel electrophoresis . Iyo inoshandiswa kuparadzanisa zvidimbu zveDNA zvichienderana nehukuru uye mutero. Zvidimbu izvi zvinobva zvave zvichinyatsoonekwa nekushandisa dhayi kana ma radioisotopes.
The Evolution
Sezvo kuwanikwa kwePCR, DNA polymerases imwe kunze kweTaq yekutanga yawanikwa. Mimwe yeiyi iri nani "kuverenga" kana kuti yakanyatsogadzikana pamatambo akakwirira, nokudaro nekuvandudza zvakananga zvePCR uye kuderedza zvikanganiso kubva pakuiswa kweDNTP isina kururama.
Zvimwe zvakasiyana-siyana zvePCR zvakagadzirirwa zvikumbiro zvakashandiswa uye zvino zvinoshandiswa nguva dzose mumaserocular genetic laboratories. Mamwe aya ndea Real-Time PCR uye Reverse-Transcriptase PCR. Kuwanikwa kwePCR kwave kuchiitawo kukurudzirwa kweDNA kushambadzira, DNA zvinyorwa zvinyorwa uye mamwe maitiro emakemikari.