Ukulandelelana kwe-DNA kuxhomekeke ekubeni sikwazi ukusebenzisa i- gel electrophoresis ukuhlukanisa imida ye-DNA eyahlukileyo ngobukhulu njengento encinane enye.
DNA Sequencing
Ngasekupheleni kwee-1970, ezimbini iindlela zokulandelela i-DNA yee-molecule zeDNA ezide zenziwe. Le ndlela yayiyi- Sanger (okanye i-videoxy) nendlela kunye ne- Maxam-Gilbert (indlela yokwenza imichiza). Indlela ye-Maxam-Gilbert isekelwe kwi-nucleotide-specific cleavage ngamakhemikhali kwaye isetyenziselwa ukulandelelanisa i-oligonucleotides Indlela yeSanger isetyenziswa ngokuqhelekileyo kuba ibonakaliswe lula ukuba isebenzise, kwaye, ngokufika kwe-PCR kunye nokuzenzekelayo kwindlela, isetyenziswe lula kwiinkalo ezide ze-DNA kubandakanya ezinye izakhi zofuzo. Le ndlela isekelwe ekutyunjweni kwekhenketho ngama-videoxynucleotides ngexesha lokuphendula kwe-PCR.
Indlela yeSanger
Kwiindlela zeSanger, i-DNA strand ehlaziywa isetyenziswa njengetemplate kunye ne-DNA polymerase isetyenzisiweyo, kwi-PCR ukusabela, ukuvelisa imida eqhotyoshelweyo isebenzisa izibhengezo.
Iintlobo ezahlukeneyo zokusabela kwe-PCR ezahlukeneyo zilungiselelwe, nganye iqulethe ipesenti ethile ye-videoxynucleoside triphosphate (ddNTP) efana nenye yeenucleotides ezine (ATP, CTP, GTP okanye iTTP). I-synthetic ye-DNA entsha iyaqhubeka kude kubekho enye yezi analogs ezibandakanyiweyo, ngelo xesha i-strand ihamba ngokukhawuleza.
Impendulo nganye ye-PCR iya kugqiba iqulethe umxube wobude obude be-DNA, zonke eziphela nge-nucleotide eyayibizwa ngokuba yi-video ebizwa ngokuba yimpendulo. I-gel electrophoresis isetyenziselwa ukwahlula imida yeendlela ezithintekayo, kwiindlela ezine ezihlukeneyo, kwaye zichane ngokulandelelana kwethemplate yangaphambili ngokusekelwe kukuthi ubude bemiqolo buphela kukuphi i-nucleotide.
Kwi-automated Sanger reaction, i-primers isetyenzisiweyo ezibhalwe ngeempawu ezine ezahlukeneyo zefluorescent. Iimpendulo ze-PCR, phambi kwee-nucleotide ze-video zohluhlu, zenziwa njengoko kuchazwe ngasentla. Nangona kunjalo, ngokulandelayo, imixube emine yokusabela iyahlanganiswa kwaye isetyenziswe kwindlela enye ye-gel. Umbala weqhekeza ngalunye ufunyenwe usebenzisa i-laser bhamera kwaye ulwazi luqokelelwe yikhompyutheni eyenza i-chromatogram ebonisa imibala embala ngamnye, apho ulandelelwano lwe-DNA olulandelelanayo lunokumiselwa khona.
Ngokuqhelekileyo, indlela yokulandelela ngokuzenzekelayo ichanekile kuphela ukulandelelana ukuya kufikelela kuma-700 ukuya ku-800 amabini amabini. Nangona kunjalo, kunokwenzeka ukufumana ukulandelelana okupheleleyo kweezityalo zegciwane kwaye, ngokwenene, i-genomes epheleleyo, usebenzisa iindlela zokuziphatha ezinjenge-Primer Walking kunye ne-Shotgun yokulandelana.
Kwi- Primer Walking , isabelo esisebenzayo segeni enkulu lilandelelwano usebenzisa indlela yeSanger. Iziqalo ezintsha ziveliswa kwinqanaba elithembekileyo lokulandelelana kwaye lisetyenziselwa ukuqhubeka nokulandelelanisa isabelo somthi owawuphelelwe luhlu lweempendulo zangaphambili.
I-Shotgun yokulandelana iquka ngokukhawuleza ukusika i-DNA ingxenye yentshisekelo kumacandelo amaninzi afanelekileyo (alawulwayo), ulandelelanisa isiqephu ngasinye, kwaye uhlele iziqwenga ngokusekelwe ekulandeleni ukulandelana. Olu buchule lenziwe lula ngokusetyenziswa kwesofthiwe yekhompyutheni yokulungiselela iziqwenga ezigqithisiweyo.