DNA inotarisa inoenderanawo nekukwanisa kwatinoita gel electrophoresis kuparadzanisa michina yeDNA iyo yakasiyana nekukura kuburikidza nediki shoma rimwe chete.
DNA Sequencing
Mukupera kwema1970, maDNA maviri ekugadzirisa maitiro ekureba DNA molecules akaumbwa. Idzi dzaiva Sanger (kana dideoxy) nzira uye Maxam-Gilbert (chemical cleavage) nzira. Nzira yaMaxam-Gilbert inobva pakuchengetedzwa kwekodotide nekemikemikari uye inonyatsoshandiswa kuenzanisa oligonucleotides (mafupi emapurymondi epoymide, kazhinji ari maduku kudarika makumi maviri akaenzana-maviri murefu). Nzira yeSanger ndiyo inowanzoshandiswa nokuti yakaratidza kuti zviri nyore nyore kushandisa, uye, nekuuya kwaPCR uye kushandiswa kwemaitiro, zvinoshandiswa zviri nyore kumakumbo marefu eDNA kusanganisira mamwe mazamu ega. Iyi nzira yakagadzirwa neketani yakagadziriswa nedideoxynucleotides panguva yePCR elongation reactions.
Sanger Method
Munzira yeSanger, DNA rondedzero inoshandiswa inoshandiswa se template uye DNA polymerase inoshandiswa, mumusangano wePCR, kuti iite michina yepamusoro inoshandisa mashizha.
Zvimwe zvakasiyana-siyana zvePCR zvinosanganiswa mitsva zvakagadzirirwa, imwe neimwe inenge imwe yezana yeexyxynucleoside triphosphate (ddNTP) yakafanana kune imwe yemachina nucleotides (ATP, CTP, GTP kana TTP). Shanduro yeDNA itsva inoenderera mberi inoenderera mberi kusvikira imwe yezvifananidzi izvi ichinyorwa, panguva iyo chidimbu chisati chakatora.
Zvose zveCPR zvinopedzisira zvine musanganiswa wehutano hwakaenzana hwezvidzidzo zveDNA, zvose zvinoguma ne nucleotide iyo yaiva mavhidhiyo akanyorwa maitiro akadaro. Gel electrophoresis inoshandiswa kuparadzanisa michina yemagadziro mana, mumigwagwa mina yakasiyana, uye inotarisa kuenzaniswa kwetemplate yepakutanga inotarirwa kuti urefu hwakareba hupi hunoguma neiyo nucleotide.
Muchigadziro cheSanger chekuita, zvinyorwa zvinoshandiswa izvo zvakanyorwa nezvine mavara mana akasiyana-siyana e-fluorescent tags. Maitiro ePCR, muhupo hwemamwe maharadioxy nucleotides, anoitwa sezvinotsanangurwa pamusoro apa. Kunyange zvakadaro, zvinotevera, iyo miviri inoshandiswa inogadziriswa inosanganiswa uye inoshandiswa kune imwe nzira yejeri. Muvara wezvimwe zvidimbu zvinowanikwa uchishandiswa sarudzo uye iyo mashoko inounganidzwa nekombiyuta inogadzira chromatograms inoratidza mapuranga emuchindwe woga woga, kubva pane iyo DNA yekutevedzera inoenderana nayo inogona kugadziriswa.
Kazhinji, iyo nzira yakagadziriswa yekutsanangurira inongori yakarurama yekutevedzana kusvika kune inenge inosvika 700-800 mabhesi maviri murefu. Zvisinei, zvinokwanisika kuwana zvizere zvakakwana zvemajeni makuru uye, chaizvoizvo, zvizere zvema genomes, uchishandisa nzira dzekutsika-dzakachenjera dzakadai sa Primer Walking uye Shotgun sequencing.
MuPermer Walking , chikamu chinoshanda chejena guru chinosanganiswa kushandisa nzira yeSanger. Zvitsva zvitsva zvinoumbwa kubva kune chikamu chakatendeka chekuenzanisa uye chakashandiswa kupfuurira kuenzanisa chikamu chejeni chakanga chiri kunze kwehutano hwekuita kwepakutanga.
Shotgun sequencing inosanganisira kuparadzanisa DNA chikamu chechidimbu muzvikamu zvakakosha zvakakwana (zvinotarisirwa) zvakakosha zvakapetwa, kurongedza chikamu chimwe nechimwe, uye kuronga zvidimbu zvichienderana nekugoverana kwekugoverana. Iyi nzira yakagadziridzwa nyore nekushandiswa kwemapurogiramu epakombiyuta pakuronga zvidimbu zvakaputika.