Ulandelelwano lwe-genomic yeentsholongwane ezininzi sele ziyaziwa.I-Nucleic acid probes ezingamacandelo amafutshane e-DNA eyilelwe ukuxubana kunye ne-DNA yentsholongwane ehambelanayo okanye amacandelo e-RNA.I-polymerase chain reaction (PCR) bubuchule obusebenza ngakumbi bokubona intsholongwane.Iindlela zokuxilonga ezikumgangatho ophezulu ziye zaphuhliswa kutsha nje.
A. Nucleic acid hybridization ubuchule
I-Nucleic acid hybridization, ingakumbi ibandakanya ukubhula okusemazantsi (eMazantsi) kunye nokuvuthulula okuMantla (eMantla), yindlela entsha ephuhlayo ngokukhawuleza kwinkalo yoxilongo lwentsholongwane.Ingqiqo yovavanyo lwe-hybridization kukusebenzisa amacandelo amafutshane e-DNA (ebizwa ngokuba "yiprobe") eyenzelwe ukudibanisa kunye ne-virus ye-DNA okanye i-RNA segment.Ngokufudumeza okanye unyango lwe-alkaline, i-DNA edibeneyo ephindwe kabini okanye i-RNA ihlulwe ibe yimicu enye kwaye emva koko ifakwe kwinkxaso eqinile.Emva koko, i-probe yongezwa kwaye ixutywe kunye ne-DNA ekujoliswe kuyo okanye i-RNA.Njengoko i-probe ibhalwe nge-isotope okanye i-nuclide engekho-radioactive, i-DNA ekujoliswe kuyo okanye i-RNA inokubonwa nge-autoradiography okanye nge-biotin-avidin system.Kuba uninzi lwejenomes yentsholongwane sele yenziwe yaza yacwangciswa, inokubhaqwa kusetyenziswa ulandelelwano oluthile lwentsholongwane njenge probes kumzekelo.Okwangoku, iindlela zokuxutywa zibandakanya: i-dot blot, in situ hybridization kwiiseli, i-DNA blotting (DNA) (i-Southern blot) kunye ne-RNA blotting (RNA) (i-Northern blot).
B.PCR Technology
Kwiminyaka yakutshanje, uthotho lweendlela ze-in vitro nucleic acid amplification ziye zaphuhliswa ngokusekwe kwi-PCR, ukuvavanya iintsholongwane ezingenaluvelwano okanye ezingalinywayo.I-PCR yindlela enokuthi idibanise ulandelelwano oluthile lwe-DNA nge-in vitro polymerase reaction.Inkqubo ye-PCR ibandakanya umjikelezo we-thermal wamanyathelo amathathu: i-denaturation, i-annealing, kunye nokwandiswa Kwiqondo lokushisa eliphezulu (93℃ ~ 95℃), i-DNA ephindwe kabini ihlukaniswe ibe yimicu emibini ye-DNA;emva koko kubushushu obuphantsi (37℃ ~ 60 ℃), ezimbini ezidityanisiweyo ze-nucleotide primers anneal ukuya kumacandelo e-DNA ancedisayo;kanti kwiqondo lobushushu elifanelekileyo le-Taq enzyme (72℃), ukuhlanganiswa kwamatyathanga eDNA amatsha aqala kwiprimer 3'end kusetyenziswa i-DNA encedisanayo njengeetemplates kunye neenucleotides enye njengemathiriyeli.Ngoko emva komjikelo ngamnye, ikhonkco elinye leDNA linokwandiswa libe ngamatyathanga amabini.Ukuphindaphinda le nkqubo, ikhonkco ngalinye le-DNA elenziwe kumjikelo omnye lingasetyenziswa njenge template kumjikelo olandelayo, kwaye inani lamatyathanga e-DNA liphindwe kabini kumjikelo ngamnye, oku kuthetha ukuba ukuveliswa kwe-PCR kwandiswe kwisantya selogi ye-2n.Emva kwe-25 ukuya kwi-30 imijikelezo, ukuveliswa kwe-PCR ichongiwe nge-electrophoresis, kwaye iimveliso ezithile ze-DNA zinokubonwa phantsi kokukhanya kwe-UV (254nm).Ngenzuzo yayo ethile, uvakalelo, kunye nokulula, i-PCR yamkelwe kuxilongo lwezonyango lwezifo ezininzi zentsholongwane ezinjenge-HCV, i-HIV, i-CMV, kunye ne-HPV.Njengoko i-PCR inovakalelo kakhulu, inokubona intsholongwane ye-DNA kwinqanaba le-fg, utyando kufuneka lwenziwe ngononophelo olukhulu ukunqanda ubuxoki.Ukongeza, iziphumo ezilungileyo kuvavanyo lwe-nucleic acid ayithethi ukuba kukho intsholongwane ephilayo eyosulelayo kwisampulu.
Ngokusetyenziswa okubanzi kobuchule be-PCR, ubuchule obutsha kunye neendlela ziyaphuhliswa ngokusekwe kubuchule be-PCR ngeenjongo ezahlukeneyo zovavanyo.Umzekelo, ixesha lokwenyani lobungakanani bePCR inokubona umthamo wentsholongwane egazini;kwi-situ PCR isetyenziselwa ukuchonga usulelo lwentsholongwane kwiithishu okanye kwiiseli;I-PCR efakwe kwindlwane inokunyusa ubume be-PCR.Phakathi kwabo, ixesha lokwenyani le-PCR liphuhliswe ngokukhawuleza.Uninzi lweendlela ezintsha, ezinje nge-TaqMan hydrolysis probe, hybridization probe, kunye ne-molecular beacon probe, zidityanisiwe kubuchule bexesha lokwenyani lobungakanani bePCR, obusetyenziswa ngokubanzi kuphando lwezonyango.Ngaphandle kokuchonga umthamo wentsholongwane egazini kulwelo lomzimba wesigulana ngokuchanekileyo, le ndlela isenokusetyenziswa ukufumanisa inguqulelo ekwaziyo ukumelana neziyobisi.Ke ngoko, i-PCR yexesha lokwenyani isetyenziswa ikakhulu kuvavanyo lwesiphumo sonyango kunye novavanyo lokunyamezela ichiza.
C. Ukufunyanwa okuphezulu kwe-viral nucleic acids
Ukuhlangabezana neemfuno zokuxilongwa ngokukhawuleza kwezifo ezitsha ezosulelayo, iindlela ezahlukeneyo zokufumanisa iziphumo eziphezulu, ezifana ne-DNA chips (DNA), zisekiwe.Kwiichips zeDNA, iiprobe ezithile ziyadityaniswa kwaye zincanyathiselwe kwiitshiphusi zesilicon ezincinci kuxinaniso oluphezulu kakhulu ukwenza iDNA probe microarray (DNA) enokuthi ixutywe ngesampulu.Umqondiso wokuxubana unokubonwa nge-microscope edibeneyo okanye i-laser scanner kwaye iqhutywe ngakumbi yikhompyutheni kunye neseti enkulu yedatha malunga nejeneloli eyahlukeneyo inokufumaneka.Kukho iindidi ezimbini ze-DNA chip."I-chip ye-synthesis" ilandelayo: i-oligonucleotides ethile yenziwe ngokuthe ngqo kwiichips.Enye yi-DNA pool chip.Iijini ezidibeneyo okanye iimveliso zePCR ziprintwa ngocwangco kwisilayidi.Uncedo lwetekhnoloji yetshiphu ye-DNA kukubhaqwa kwangaxeshanye ubuninzi obukhulu bokulandelelana kwe-DNA.Inguqulelo yamva nje yetshiphu yokufumanisa i-pathogen inokuchonga ngaphezulu kwe-1700 yeentsholongwane zabantu ngaxeshanye.Itekhnoloji ye-DNA chip yasombulula iingxaki zeendlela ze-nucleic acid hybridization kwaye inezicelo ezibanzi kakhulu ekuxilongweni kwentsholongwane kunye nophononongo lwe-epidemiological.
Ixesha lokuposa: Dec-23-2020