A mara usoro mkpụrụ ndụ ihe nketa nke ọtụtụ nje.Nchọpụta nucleic acid bụ akụkụ dị mkpirikpi nke DNA emebere ka ijikọ DNA ma ọ bụ RNA viral agbakwunyere.Mmeghachi omume yinye polymerase (PCR) bụ usoro na-arụ ọrụ nke ọma maka nchọpụta nje.Emepụtala ụzọ nchọpụta dị elu n'oge na-adịbeghị anya.
A. Nucleic acid hybridization Usoro
Nucleic acid hybridization, tumadi gụnyere Southern blotting (Southern) na Northern blotting (Northern), bụ ngwa ngwa na-emepe emepe ọhụrụ usoro na nje diagnostic ubi.Ebumnuche nke nyocha ngwakọ bụ iji akụkụ dị mkpirikpi nke DNA (nke a na-akpọ "nyocha") emebere iji mejupụta DNA ma ọ bụ RNA viral agbakwunyere.Site na ikpo ọkụ ma ọ bụ ọgwụgwọ alkaline, DNA ma ọ bụ RNA nwere eriri abụọ na-ekewa n'ime otu eriri wee mee ka ọ ghara imegharị ya na nkwado siri ike.Mgbe nke ahụ gasịrị, a na-agbakwunye nyocha na ngwakọ ya na DNA ma ọ bụ RNA e lekwasịrị anya.Dị ka a na-akpọ nyocha ahụ na isotope ma ọ bụ nuclide na-abụghị nke redio, a pụrụ ịchọpụta DNA ma ọ bụ RNA e lekwasịrị anya site na autoradiography ma ọ bụ site na usoro biotin-avidin.Ebe ọ bụ na ọtụtụ n'ime mkpụrụ ndụ ihe nketa viral ka ejirila cloned na usoro, enwere ike ịchọpụta ha site na iji usoro nje kpọmkwem dị ka nyocha dị na ụdị.Ka ọ dị ugbu a, usoro nke ngwakọ gụnyere: ntụpọ ntụpọ , n'ụdị hybridization na mkpụrụ ndụ, DNA blotting (DNA) (Southern blot) na RNA blotting (RNA) (Northern blot).
B.PCR teknụzụ
N'ime afọ ndị na-adịbeghị anya, e mepụtala usoro nke in vitro nucleic acid amplification technology dabere na PCR, iji nwalee nje ndị na-adịghị ahụkebe ma ọ bụ ndị a na-apụghị ịkụ mkpụrụ.PCR bụ usoro nke nwere ike ijikọta usoro DNA kpọmkwem site na mmeghachi omume in vitro polymerase.Usoro nke PCR na-agụnye usoro okpomọkụ nke usoro atọ: denaturation , annealing , and extension Na elu okpomọkụ (93 ℃ ~ 95 ℃), DNA nke nwere eriri abụọ na-ekewa n'ime eriri DNA abụọ;mgbe ahụ, na obere okpomọkụ (37 ℃ ~ 60 ℃), abụọ synthesized nucleotide primers anneal na-arụkọ DNA akụkụ;ebe na okpomọkụ kwesịrị ekwesị maka Taq enzyme (72 ℃), njikọ nke agbụ DNA ọhụrụ na-amalite site na primer 3'end site na iji DNA nkwado dị ka ndebiri na otu nucleotides dị ka ihe.Ya mere, mgbe okirikiri nke ọ bụla gasịrị, a pụrụ ịgbanye otu yinye DNA ka ọ bụrụ ụdọ abụọ.N'ịmeghachi usoro a, a na-eji yinye DNA ọ bụla arụpụtara n'otu okirikiri mee ihe dị ka ndebiri na okirikiri na-esote, na ọnụọgụ DNA na-agbakọ okpukpu abụọ na okirikiri nke ọ bụla, nke pụtara na mmepụta PCR na-abawanye na ọsọ ọsọ 2n.Mgbe usoro 25 ruo 30 gasịrị, a na-achọpụta mmepụta PCR site na electrophoresis , a pụkwara ịhụ ngwaahịa DNA kpọmkwem n'okpuru ìhè UV (254nm).Maka uru nke nkọwa ya, uche, na ịdị mma, PCR ka anabatara na nyocha ụlọ ọgwụ nke ọtụtụ nje virus dị ka HCV, HIV, CMV, na HPV.Dị ka PCR na-enwe mmetụta nke ukwuu, ọ nwere ike ịchọpụta DNA nje na ọkwa fg, a ga-arụ ọrụ ahụ nke ọma iji zere ezigbo ụgha.Na mgbakwunye, nsonaazụ dị mma na nnwale nucleic acid apụtaghị na nje na-efe efe dị na nlele ahụ.
Site na ngwa ngwa nke usoro PCR, a na-emepụta usoro na ụzọ ọhụrụ dabere na usoro PCR maka ebumnuche ule dị iche iche.Dị ka ọmụmaatụ, ezigbo oge quantitative PCR nwere ike ịchọpụta ibu nje;na ebe a na-eji PCR chọpụta ọrịa nje na anụ ahụ ma ọ bụ sel;PCR akwụghị ụgwọ nwere ike ịbawanye nkọwapụta nke PCR.N'ime ha, ezigbo oge ọnụọgụ PCR emeputala ngwa ngwa.Ọtụtụ usoro ọhụụ, dị ka nyocha TaqMan hydrolysis, nyocha hybridization, na nyocha mgbama molekụla, ka ejikọtala na usoro PCR n'ezie, nke a na-ejikarị na nyocha ụlọ ọgwụ.E wezụga ịchọpụta ibu nje dị n'ime ahụ ndị ọrịa n'ụzọ ziri ezi, a pụkwara iji usoro a chọpụta mutant na-anabata ọgwụ.Ya mere, a na-etinye PCR n'ezie n'ụzọ zuru ezu na nyocha mmetụta ọgwụgwọ yana nleba anya nnabata ọgwụ.
C. Nchọpụta dị elu nke nucleic acid viral
Iji gboo mkpa maka nyocha ngwa ngwa nke ọrịa na-efe efe ọhụrụ, ụzọ dị iche iche na-achọpụta ihe dị elu, dị ka ibe DNA (DNA), ehiwela.Maka ibe DNA, a na-arụkọ ọrụ nyocha akọwapụtara ma tinye ya na obere silicon ibe njupụta dị oke elu iji mepụta DNA nyocha microarray (DNA) nke enwere ike iji nlele mee ngwakọ.Enwere ike iji microscope confocal ma ọ bụ nyocha laser gosipụta ihe mgbaàmà nke hybridization wee nwee ike ịhazi ya site na kọmpụta ọzọ wee nweta nnukwu data gbasara mkpụrụ ndụ ihe nketa dị iche iche.Enwere ụdị mgbawa DNA abụọ."Mgbawa njikọ" dị ka ndị a: a na-emepụta oligonucleotides kpọmkwem na ibe.Ọzọ bụ DNA ọdọ mmiri mgbawa.A na-ebipụta mkpụrụ ndụ ihe nketa cloned ma ọ bụ ngwaahịa PCR n'usoro na slide ahụ.Uru nke teknụzụ mgbawa DNA bụ nchọpụta n'otu oge nke nnukwu usoro DNA.Ụdị mgbawa nchọpụta pathogen ọhụrụ kachasị ọhụrụ nwere ike ịchọpụta ihe karịrị nje mmadụ 1700 n'otu oge.Teknụzụ mgbawa DNA doziri nsogbu nke ụzọ hybridization nke nucleic acid ọdịnala ma nwee ngwa sara mbara na nyocha nje na nyocha ọrịa.
Oge nzipu: Dec-23-2020