Ukulandelana kwe-genomic kwamagciwane amaningi sekwaziwa.Ama-Nucleic acid probes amasegimenti amafushane e-DNA aklanyelwe ukuhlanganisa amasegimenti egciwane le-DNA noma i-RNA ehambisanayo.I-polymerase chain reaction (PCR) iyindlela esebenza kahle kakhulu yokuthola amagciwane.Izindlela zokuxilonga ezisezingeni eliphezulu zakhiwe muva nje.
A. I-Nucleic acid hybridization technique
I-Nucleic acid hybridization, ikakhulukazi okuhlanganisa i-Southern blotting(iNingizimu) kanye ne-Northern blotting (eNyakatho), kuyindlela entsha ethuthuka ngokushesha emkhakheni wokuxilongwa kwegciwane.Isizathu sokuhlolwa kwe-hybridization ukusebenzisa izingxenye ezimfushane ze-DNA (ezibizwa ngokuthi “i-probe”) eklanyelwe ukuhlanganisa nge-DNA yegciwane ehambisanayo noma izingxenye ze-RNA.Ngokushisisa noma ukwelashwa nge-alkaline, i-DNA enemicu ekabili noma i-RNA ihlukaniswa ibe imicu eyodwa bese inganyakazi ekusekelweni okuqinile.Ngemuva kwalokho, i-probe iyengezwa futhi ihlanganiswe ne-DNA eqondiwe noma i-RNA.Njengoba uphenyo lubhalwe nge-isotopu noma i-nuclide engasebenzisi umsakazo, i-DNA eqondiwe noma i-RNA ingatholwa nge-autoradiography noma ngohlelo lwe-biotin-avidin .Njengoba iningi lama-virus genome enziwe aklanywa futhi alandelana, angatholwa kusetshenziswa ukulandelana okuqondene negciwane njengama-probe kusampula.Njengamanje, izindlela zokuhlanganisa zihlanganisa: i-dot blot , i-in situ hybridization kumaseli , i-DNA blotting(DNA) (i-Southern blotting) kanye ne-RNA blotting(RNA) (i-Northern blot).
Ubuchwepheshe be-B.PCR
Eminyakeni yamuva nje, kuye kwasungulwa uchungechunge lwezindlela zokukhulisa i-in vitro nucleic acid ezisekelwe ku-PCR, ukuhlola amagciwane angenazwelo noma angenakutshalwa.I-PCR iyindlela engahlanganisa ukulandelana kwe-DNA ethile nge-in vitro polymerase reaction.Inqubo ye-PCR ihlanganisa umjikelezo oshisayo wezinyathelo ezintathu: i-denaturation, annealing, kanye nesandiso Emazingeni okushisa aphezulu (93℃~95℃), i-DNA enemicu ekabili ihlukaniswa ibe imicu emibili ye-DNA eyodwa;bese kuthi izinga lokushisa eliphansi (37℃~60℃), iziqalo ezimbili ze-nucleotide ezihlanganisiwe zingene ezingxenyeni ezihambisanayo ze-DNA;kanti ezingeni lokushisa elifanele le-enzyme ye-Taq (72℃), ukuhlanganiswa kwamaketanga e-DNA amasha kuqala kusukela ku-primer 3'end kusetshenziswa i-DNA ehambisanayo njengezifanekiso kanye nama-nucleotide awodwa njengento esetshenziswayo.Ngakho ngemva komjikelezo ngamunye, iketango elilodwa le-DNA lingakhuliswa libe ngamaketanga amabili.Ukuphinda le nqubo, iketango le-DNA ngalinye elihlanganiswe emjikelezweni owodwa lingasetshenziswa njengesifanekiso kumjikelezo olandelayo, futhi inani lamaketanga e-DNA liphindwe kabili emjikelezweni ngamunye, okusho ukuthi ukukhiqizwa kwe-PCR kukhuliswa ngesivinini selogi engu-2n.Ngemuva kwemijikelezo engama-25 kuye kwengama-30, ukukhiqizwa kwe-PCR kukhonjwa nge-electrophoresis, futhi imikhiqizo ethile ye-DNA ingabonwa ngaphansi kokukhanya kwe-UV (254nm).Ngenzuzo yayo yokucacisa, ukuzwela, kanye nokuba lula, i-PCR yamukelwe ekuxilongweni komtholampilo kwezifo eziningi ezibangelwa amagciwane njenge-HCV, i-HIV, i-CMV, ne-HPV.Njengoba i-PCR izwela kakhulu, ingathola i-virus ye-DNA ezingeni le-fg, ukuhlinzwa kufanele kwenziwe ngokucophelela ukuze kugwenywe ukuthi unamanga.Ngaphezu kwalokho, umphumela omuhle ekuhlolweni kwe-nucleic acid awusho ukuthi kunegciwane elithelelanayo eliphilayo kusampula.
Ngokusetshenziswa okubanzi kwendlela ye-PCR, amasu amasha nezindlela ziyathuthukiswa ngokususelwe kubuchule be-PCR ngezinjongo ezihlukene zokuhlola.Isibonelo, i-PCR yobuningi besikhathi sangempela ingathola umthamo wegciwane;in situ PCR isetshenziselwa ukukhomba ukutheleleka ngegciwane ezicutshini noma kumaseli;I-PCR evalelwe ingakhuphula ukucaciswa kwe-PCR.Phakathi kwazo, i-PCR yobuningi besikhathi sangempela yakhiwe ngokushesha okukhulu.Amasu amaningi amasha, anjenge-TaqMan hydrolysis probe, hybridization probe, kanye ne-molecular beacon probe, ahlanganiswe abe yinqubo ye-PCR yobuningi besikhathi sangempela, esetshenziswa kabanzi ocwaningweni lomtholampilo.Ngaphandle kokuhlonza umthamo wegciwane egazini oketshezini lomzimba weziguli ngokunembile, le ndlela ingase futhi isetshenziselwe ukuthola i-mutant ebekezelela izidakamizwa.Ngakho-ke, i-PCR yobuningi besikhathi sangempela isetshenziswa kakhulu ekuhlolweni komphumela wokwelapha kanye nokubhekwa kokubekezelela izidakamizwa.
C. Ukutholwa okuphezulu kwe-viral nucleic acids
Ukuze kuhlangatshezwane nezidingo zokuxilonga ngokushesha kwezifo ezintsha ezithathelwanayo eziqhamukayo, kusungulwe izindlela ezihlukahlukene zokuthola umphumela omkhulu, njengama-chips e-DNA(DNA).Kuma-chips e-DNA, ama-probe athile ayahlanganiswa futhi anamathiselwe kuma-silicon chips amancane anokuminyana okuphezulu kakhulu ukuze akhe i-DNA probe microarray (DNA) engahlanganiswa ngesampula.Isignali yokuhlanganisa ingathwetshulwa ngesibonakhulu esihlanganayo noma isithwebuli se-laser futhi iqhutshekwe kusetshenziswa ikhompuyutha futhi kutholakale idatha enkulu ephathelene nezakhi zofuzo ezihlukene.Kunezinhlobo ezimbili ze-DNA chip.I-"synthesis chip" injengoba ilandelayo: ama-oligonucleotide athile ahlanganiswa ngqo kuma-chips.Enye i-DNA pool chip.Izakhi zofuzo ezihlanganisiwe noma imikhiqizo ye-PCR iphrintwa ngokuhlelekile kusilayidi.Inzuzo yobuchwepheshe be-DNA chip ukutholwa ngasikhathi sinye kwenani elikhulu lokulandelana kwe-DNA.Inguqulo yakamuva ye-chip yokuthola i-pathogen ingakwazi ukukhomba amagciwane abantu angaphezu kuka-1700 ngesikhathi esisodwa.Ubuchwepheshe be-DNA chip buxazulule izinkinga zezindlela zendabuko ze-nucleic acid hybridization futhi busebenza kakhulu ekuxilongweni kwegciwane kanye nocwaningo lwe-epidemiological.
Isikhathi sokuthumela: Dec-23-2020