I-gene cloning yinto yokwenza iikopi, okanye i-clones, yesigidi esisodwa. Emva kokuba i-gene ichongiwe, iifayile zingasetyenziselwa kwiindawo ezininzi zophando lwezolimo kunye nezoshishino. Ubunjineli be-Genetic yinkqubo yokwenza i-cloning gene to new organisms okanye ukutshintsha i-DNA ngokulandelelana ukuguqula umkhiqizo weprotheni. Ubunjineli be-Genetic buxhomekeke ekusebenzeni kwethu ukwenza le miqathango ebalulekileyo.
01 Polymerase Chain Reaction
02 Ukulungiswa kweezyzymes
Ukufunyanwa kwee- enzyme ezibizwa ngokuba yi-restriction endonucleases kubalulekile kubunjineli beprotein . Ezi enzyme zinqumle i-DNA kwiindawo ezithile ngokulandelelana kwe-nucleotide. Amakhulu ahlukeneyo e- restriction enzymes , anokukwazi ukusika i-DNA kwisayithi esicacileyo, sele ikhutshwe kwiintlobo ezahlukeneyo zebhaktheriya. I-DNA enqunywe nge-enzyme yokuthintela ivelisa amaninzi amancinci amancinci, ahlukeneyo. Ezi zinokuhlukaniswa usebenzisa i-gel electrophoresis okanye i-chromatography.
03 Electrophoresis
I-DNA ecocekileyo kwinkcubeko yeselula, okanye ukuyinquma ngokusebenzisa i-restriction enzymes ayinakusetyenziswa kakhulu xa singenakukwazi ukubona i-DNA - oko kukuthi, fumana indlela yokujonga ukuba ingaba i-extract yakho iqukethe nayiphi na into Ndiyinqumle. Enye indlela yokwenza oku yi-gel electrophoresis. Iigels zisetyenziselwa iinjongo ezahlukeneyo, ukusuka ekubukeni i-DNA esinqumle ukufumana ukufakwa kwe-DNA kunye neenkqonkqo.
04 Hlanganisa ezimbini zeeDNA
Kuphando lwezofuzo, kubakho imfuneko ukudibanisa iimbumba ezimbini okanye ngaphezulu kweDNA, ukudala i-strand recombinant, okanye uvale i-strand ye-circular eye isinqunywe kunye ne-restriction enzymes. Ii-enzymes ezibizwa ngokuba yi-DNA ligases zingenza izibophelelo ezidibeneyo phakathi kwamaketanga e-nucleotide. I-enzymes i-DNA polymasease I kunye ne-polynucleotide kinase nazo zibalulekile kule nkqubo, ukuzaliswa kwezikhefu okanye i-phosphorylating ye-5's ends, ngokulandelanayo.
05 Ukukhethwa kwe-DNA yokuzicoca ngokuncinci
Izicatshulwa ezincinci zeDNA ezingenxalenye yegciwane lesibindi, kodwa zikwazi ukuziphindaphinda, zibizwa ngokuba yi-plasmids. Iiplasmids zisoloko zisetyenziselwa njengezitshixo zokuthutha iigeni phakathi kwama-microorganms. Kwi-biotechnology, xa i-gene ye-interest ikhutshwe kwaye i-gene kunye ne-plasmid zinqunywe ngumda wokukhutshwa kwe-enzymes, zixutywe kunye kunye nokwenza into eyaziwayo njenge-DNA ephindaphindiweyo. Intsholongwane (i-bacteriophage) I-DNA ingasetyenziselwa njengemvolonti, njengokuba isenza i-cosmids, i-plasmids ephindaphindiweyo equkethe i-bacteriophage genes.
06 Indlela yokuhambisa iVector kwiSeli yeSigqeba
Inkqubo yokudlulisa izinto eziphathekayo kwi-vector efana ne-plasmid, kumaseli amatsha amatsha, kuthiwa nguqulelo. Le nqubo idinga ukuba iiseli ezamkelekileyo zibonakalise utshintsho lwendalo ngumbonakalo olwenza ukuba "zikwazi" okanye zenzeke okwesikhashana kwi vector. I-Electroporation yinto enye enjalo. Inkulu iplasmid, ephantsi kwekhono elithatyathwa ngamaseli. Amacandelo amakhulu e-DNA akhethwa ngokulula ngokusebenzisa i-bacteriophage, i-retrovirus okanye enye i-vectors okanye i-cosmids ngendlela ebizwa ngokuthi ukutshintshwa. I-Phage okanye i-vectors vectors isoloko isetyenziswe kwiyeza elitsha ngokutsha kodwa zingabangela ukufakwa kwe-DNA kwiindawo zethu ze-chromosomes apho asifuni, kubangela iingxaki kunye nomhlaza.
07 Izindlela zokukhetha ii-Transgenic Organisms
Azikho zonke iiseli eziza kuthatha i-DNA ngenguqu. Kubalulekile ukuba kubekho indlela yokubona abo benzayo. Ngokuqhelekileyo, i-plasmids ithwala iigleji zokuchasana nomzimba kunye neeseli zesitenki zingakhethwa ngokubhekiselele ekubonakalisweni kwezi geni kunye nokukwazi kwabo ukukhula kwiimidiya eziqulethe i-antibiotic. Ezinye iindlela zokukhetha zixhomekeke kubukho bezinye iiprotheni ezinjenge-x-gal / lacZ , okanye iprotheni ye-fluorescence ehlaza, evumela ukhetho ngokusekelwe kumbala kunye ne-fluorescence, ngokulandelanayo.