PCR bụ teknụzụ nkwalite nucleic acid kachasị eji eme ihe ma ejiri ya mee ihe n'ihi nghọta ya na nkọwapụta ya.Agbanyeghị, PCR chọrọ denaturation thermal ugboro ugboro na enweghị ike iwepu oke nke ịdabere na ngwa na akụrụngwa, nke na-egbochi ngwa ya na nnwale ụlọ ọgwụ.

Kemgbe mmalite 1990s, ọtụtụ ụlọ nyocha amalitela ịmepụta teknụzụ nkwalite okpomọkụ mgbe niile nke na-achọghị denaturation thermal.Ugbu a, ha ewepụtala teknụzụ nkwalite isothermal na-eme ka akaghị aka, teknụzụ nnọchi isothermal amplification teknụzụ, teknụzụ nkwalite isothermal okirikiri, na ịdabere n'usoro nucleic acid.Teknụzụ nkwalite isothermal na teknụzụ ndị ọzọ. 

Loop-mediated isothermal amplification

Ụkpụrụ nkwalite ahụ dabere n'eziokwu ahụ bụ na DNA nọ n'ọnọdụ nha anya dị ike na ihe dịka 65 Celsius.Mgbe ejikọtara primer ọ bụla na ntọala ma gbasaa ruo akụkụ nkwado nke DNA nwere eriri abụọ, eriri nke ọzọ ga-ekewa ma ghọọ otu eriri.

N'oge okpomọkụ a, DNA na-eji 4 kpọmkwem primers ka ọ dabere na DNA polymerase nke na-akwagharị strand iji mee ka njikọ nke DNA na-ebugharị n'onwe ya na-ekesa n'onwe ya.

Buru ụzọ chọpụta mpaghara 6 kpọmkwem F3, F2, F1, B1, B2, B3 na mkpụrụ ndụ ebumnuche, wee chepụta primers 4 dabere na mpaghara 6 ndị a akọwapụtara (dị ka egosiri na foto dị n'okpuru):

Ihe mejuputara n'ime n'ihu (FIP) bụ F1c na F2.

Ihe mejupụtara n'ime ime azụ (BIP) bụ B1c na B2, na TTTT na-eji dị ka oghere n'etiti.

Mpụta primers F3 na B3 bụ otu n'otu n'otu mejupụtara mpaghara F3 na B3 na mkpụrụ ndụ ihe atụ.

N'ime usoro mmeghachi omume LAMP, ntinye nke primer dị n'ime bụ ugboro ugboro karịa nke mpụta.A na-ebu ụzọ jikọta primer dị n'ime ya na eriri template iji mepụta eriri agbakwunyere iji mepụta eriri abụọ DNA.Na-esote, a na-ejikọta primer dị n'èzí na eriri template iji mepụta eriri abụọ DNA.N'okpuru ọrụ nke BstDNA polymerase, a na-ewepụta eriri agbakwunyere site na primer nke ime.Mgbe ọtụtụ mmeghachi omume gasịrị, eriri mmekọ ahụ mechara mepụta otu eriri DNA nwere usoro dumbbell.

A na-eji usoro dumbbell DNA otu eriri n'onwe ya mee ihe dị ka ndebiri ka ọ na-etolite DNA ngbanwe azuokokoosisi nke nwere njedebe mepere emepe.N'ime na mpụta primers na-eduzi DNA mgbanwe azuokokoosisi-loop nhazi ka ọ na-aga n'ihu na-aga n'ihu na nchụpụ strand na mmeghachi omume ndọtị, na n'ikpeazụ na-etolite ọtụtụ akụkụ-akaghị aka nwere ogologo dị iche iche.Ngwakọta DNA.

Uru na ọghọm dị na nkwalite isothermal na-eme ka akaghị aka

Uru nke LAMP:

(1) Ọrụ nkwalite dị elu, nke nwere ike ịbawanye n'ụzọ dị irè 1-10 mbipụta nke mkpụrụ ndụ ihe mgbaru ọsọ n'ime 1h, yana arụmọrụ nkwalite bụ 10-100 ugboro karịa nke PCR nkịtị.

(2) Oge mmeghachi omume dị mkpụmkpụ, nkọwa ahụ siri ike, ọ dịghịkwa ngwá ọrụ pụrụ iche achọrọ.

Ajọ nke LAMP:

(1) Ihe ndị a chọrọ maka primers dị elu karịsịa.

(2) Enweghị ike iji ngwaahịa agbakwunyere maka cloning na usoro, mana enwere ike iji ya naanị maka ikpe.

(3) N'ihi mmetụta siri ike ya, ọ dị mfe ịmepụta ikuku aerosols, na-eme ka ọ bụrụ ihe na-ezighị ezi ma na-emetụta nsonaazụ ule.

Strand displacement amplification

Strand displacement amplification (SDA) bụ usoro mmụba DNA nke isothermal in vitro dabere na mmeghachi omume enzymatic nke mbụ onye ọkà mmụta America bụ Walker tụpụtara na 1992.

Sistemụ bụ isi nke SDA gụnyere mmachi endonuclease, DNA polymerase nwere ọrụ mbugharị eriri, ụzọ abụọ nke primers, dNTPs, na calcium na magnesium ion na sistemụ nchekwa.

Ụkpụrụ nke nkwalite ndọpụ strand na-adabere na usoro mmachi endonuclease gbanwetụrụ na kemịkalụ na nsọtụ abụọ nke DNA ebumnuche.Endonuclease na-emepe oghere dị na eriri DNA na ebe a na-amata ya, na DNA polymerase gbatịpụrụ oghere 3′ Ọgwụgwụ wee dochie eriri DNA ọzọ.

Enwere ike ijikọ otu eriri DNA dochie anya ya na primers wee gbatịa ya na eriri abụọ site na DNA polymerase.A na-emeghachi usoro a ugboro ugboro, nke mere na usoro ebumnuche na-abawanye nke ọma.

Uru na ọghọm dị na teknụzụ mmụba strand displacement

Uru nke SDA:

Nrụpụta mmụba dị elu, oge mmeghachi omume dị mkpụmkpụ, nkọwa siri ike, ọ dịghịkwa ngwá ọrụ pụrụ iche achọrọ.

Mmejọ nke SDA:

Ngwaahịa ndị ahụ abụghị otu, na ụfọdụ ngwaahịa nwere otu eriri na nke nwere okpukpu abụọ ka a na-emepụta mgbe niile na okirikiri SDA, na ọdụ ga-apụtarịrị ma ọ bụrụ na electrophoresis achọpụtara ya.

Rnkwalite okirikiri olling

Rolling Circle amplification (RCA) ka a na-atụpụta ya site n'ịbịara na usoro nke iṅomi DNA sitere na ihe ndị na-akpata ọrịa site na ịpịgharị okirikiri.Ọ na-ezo aka na iji DNA okirikiri nwere otu eriri dị ka ndebiri na okpomọkụ na-adị mgbe niile, yana DNA polymerase pụrụ iche (dị ka Phi29) ) N'okpuru ọrụ nke ịgbagharị gburugburu DNA njikọ iji nweta mmụba nke mkpụrụ ndụ ebumnuche.

Enwere ike kewaa RCA ka ọ bụrụ mmụba ahịrị na nkwuwa okwu.Ịrụ ọrụ nke RCA linear nwere ike iru 10⁵ugboro, na arụmọrụ nke exponential RCA nwere ike iru 10⁹ugboro.

Ọdịiche dị mfe, dị ka egosiri na foto dị n'okpuru ebe a, nkwalite linear na-eji naanị 1 primer, nkwuwa okwu b nwere primers 2.

Linear RCA ka a na-akpọkwa RCA otu primer.A primer na-ejikọta na DNA okirikiri ma na-agbatị ya site n'omume nke DNA polymerase.Ngwaahịa a bụ eriri otu ahịrị nwere ọnụ ọgụgụ dị ukwuu nke usoro ugboro ugboro puku ugboro n'ogologo otu loop.

Ebe ọ bụ na ngwaahịa nke RCA linear na-ejikọta ya na mmalite mmalite, nhazi dị mfe nke mgbaàmà bụ nnukwu uru.

Exponential RCA, nke a makwaara dị ka Hyper branched amplification HRCA (Hyper branched RCA), na exponential RCA, otu primer amplifies RCA ngwaahịa, nke abụọ primer hybridizes na RCA ngwaahịa na gbatịa, na nnọchi na-ama ejikọta na RCA ngwaahịa The downstream primers gbatịa strand, na ikwugharị ndọtị na nnọchi na-emepụta a dendritic ngwaahịa RCA.

Uru na ọghọm dị na mmụba okirikiri nucleic acid

Uru nke RCA:

Mmetụta dị elu, nkọwa dị mma na ọrụ dị mfe.

Mkpebi nke RCA:

Nsogbu ndabere n'oge nchọpụta mgbaàmà.N'oge mmeghachi omume RCA, nyocha mkpọchi enweghị ikesa na ndebiri DNA ma ọ bụ RNA nke nyocha enweghị oke nwere ike iwepụta ụfọdụ akara nzụlite. 

NNkwalite nke usoro ucleicacid

Nucleic acid sequence-based amplification (NASBA) bụ teknụzụ ọhụrụ emepụtara na ndabere nke PCR.Ọ bụ mmụba nucleic acid na-aga n'ihu na isothermal nke otu ụzọ primers nwere usoro nkwalite T7 na-eduzi.Nkà na ụzụ nwere ike ịkwalite template RNA ihe dị ka ugboro 109 n'ime ihe dị ka awa 2, nke bụ 1000 ugboro dị elu karịa usoro PCR na-emekarị ma ọ dịghị achọ ngwá ọrụ pụrụ iche.

Ejiri teknụzụ a mee nchọpụta ngwa ngwa nke ọrịa ozugbo ọ pụtara, na ọtụtụ ụlọ ọrụ na-eji usoro a ugbu a na ngwa nchọpụta RNA.

Ọ bụ ezie na nkwalite RNA nwekwara ike iji teknụzụ PCR reverse transcription, NASBA nwere uru nke ya: enwere ike ịme ya n'okpuru ọnọdụ okpomọkụ mgbe niile, ọ kwụsiri ike na zie ezie karịa teknụzụ PCR ọdịnala.

Mmeghachi omume dị na 41 degrees Celsius ma na-achọ AMV (avian myeloblastosis nje) reverse transcriptase, RNase H, T7 RNA polymerase na otu ụzọ primers iji wuchaa.

Usoro a gụnyere:

primer na-aga n'ihu nwere usoro nkwado nke T7.N'oge mmeghachi omume ahụ, primer na-aga n'ihu na-ejikọta eriri RNA ma AMV enzyme na-eme ka ọ bụrụ eriri DNA-RNA okpukpu abụọ.

RNase H na-agbari RNA na eriri abụọ ngwakọ ma na-ejigide DNA nwere otu eriri.

N'okpuru ọrụ nke reverse primer na enzyme AMV, a na-emepụta eriri abụọ DNA nwere usoro nkwalite T7.

N'okpuru ọrụ nke T7 RNA polymerase, a na-emecha usoro ntụgharị ma mepụta nnukwu RNA lekwasịrị anya.

Uru nke NASBA:

(1) Ihe mmalite ya nwere usoro mgbasa ozi T7, mana DNA nke abụọ nke mba ọzọ enweghị usoro nkwalite T7 na enweghị ike ịbawanyewanye ya, ya mere teknụzụ a nwere nkọwa dị elu na nghọta.

(2) NASBA na-etinye ozugbo na usoro ntụgharị ntụgharị n'ime mmeghachi omume mmụba, na-ebelata oge mmeghachi omume.

Ọdịmma nke NASBA:

(1) Akụkụ mmeghachi omume na-agbagwoju anya karị.

(2) A chọrọ ụdị enzymes atọ iji mee ka mmeghachi omume ahụ dị elu.