PCR ita ce fasahar haɓaka haɓakar acid nucleic da aka fi amfani da ita kuma ana amfani da ita sosai saboda azanci da ƙayyadaddun sa.Koyaya, PCR yana buƙatar maimaita ƙarancin zafi kuma ba zai iya kawar da iyakokin dogaro da kayan aiki da kayan aiki ba, wanda ke iyakance aikace-aikacen sa a gwajin filin asibiti.

Tun farkon shekarun 1990, dakunan gwaje-gwaje da yawa sun fara haɓaka fasahar haɓaka zafin jiki akai-akai wanda baya buƙatar ƙarancin zafi.Yanzu sun haɓaka fasahar haɓaka haɓakar isothermal madauki, madaurin maye gurbin fasahar haɓaka isothermal, fasahar ƙara girman da'irar isothermal, da dogaro da jerin nucleic acid.Isothermal haɓaka fasaha da sauran fasaha. 

Loop-tsakaici isothermal amplification

Ƙa'idar haɓakawa ta dogara ne akan gaskiyar cewa DNA yana cikin yanayin ma'auni mai ƙarfi a kusan 65 ° C.Lokacin da kowane firamare aka haɗa tushen tushe kuma aka mika shi zuwa sashin da ya dace na DNA mai madauri biyu, ɗayan layin zai rabu kuma ya zama mai ɗaci ɗaya.

A wannan zafin jiki, DNA tana amfani da ƙayyadaddun filaye guda 4 don dogara ga DNA polymerase mai juyawa don yin kirar DNA na matsuguni da kansa ya ci gaba da zagayawa.

Da farko ƙayyade takamaiman yankuna na 6 F3, F2, F1, B1, B2, B3 akan jigon manufa, sa'an nan kuma ƙirƙira ƙirar 4 dangane da waɗannan takamaiman yankuna 6 (kamar yadda aka nuna a hoton da ke ƙasa):

Na gaba na ciki (FIP) ya ƙunshi F1c da F2.

Na baya na ciki (BIP) ya ƙunshi B1c da B2, kuma TTTT ana amfani dashi azaman sarari a tsakiya.

Fuskokin waje F3 da B3 sun ƙunshi yankuna F3 da B3 akan jigon manufa.

A cikin tsarin amsa LAMP, ƙaddamar da abin da ke ciki ya ninka sau da yawa fiye da na waje.An fara haɗe firamare na ciki tare da madaidaicin samfuri don haɗa madaidaicin madauri don samar da madauri biyu na DNA.Daga baya, ana haɗe firamare na waje tare da madaidaicin samfur don samar da madauri biyu na DNA.Karkashin aikin BstDNA polymerase, ana fitar da madaidaicin madauri da aka haɗa ta hanyar firamare na ciki.Bayan jerin halayen, madaidaicin madaidaicin a ƙarshe ya samar da jigon DNA guda ɗaya tare da tsarin dumbbell.

Tsarin dumbbell DNA guda madauri kanta ana amfani da shi azaman samfuri don ci gaba da samar da tsarin DNA na madauki na wucin gadi tare da buɗaɗɗen ƙarshen.Nau'i-nau'i na ciki da na waje suna jagorantar tsarin DNA na wucin gadi mai tushe don ci gaba da ci gaba da jujjuya matsuguni da halayen haɓakawa, kuma a ƙarshe su samar da tsarin madauki da yawa tare da tsayi daban-daban.cakuda DNA.

Abũbuwan amfãni da rashin amfani na madauki-matsakaici na haɓaka isothermal

Amfanin LAMP:

(1) Babban haɓakar haɓakawa, wanda zai iya haɓaka kwafin 1-10 na jigon manufa a cikin 1h, kuma ƙarfin haɓakawa shine sau 10-100 na PCR na yau da kullun.

(2) Lokacin amsawa gajere ne, ƙayyadaddun ƙayyadaddun yana da ƙarfi, kuma babu kayan aiki na musamman da ake buƙata.

Karancin LAMP:

(1) Abubuwan buƙatun don abubuwan farawa suna da girma musamman.

(2) Ba za a iya amfani da ingantaccen samfurin don cloning da sequencing ba, amma ana iya amfani da shi kawai don hukunci.

(3) Saboda tsananin hankalinsa, yana da sauƙi don samar da iska mai iska, yana haifar da halayen ƙarya kuma yana shafar sakamakon gwajin.

Strand ƙaura haɓakawa

Strand displacement amplification (SDA) dabara ce ta in vitro isothermal ƙarawa DNA da ta dogara da halayen enzymatic wanda masanin Ba'amurke Walker ya fara gabatarwa a cikin 1992.

Tsarin asali na SDA ya haɗa da ƙuntatawa endonuclease, DNA polymerase tare da aikin ƙaura, nau'i-nau'i biyu na al'ada, dNTPs, da calcium da magnesium ions da tsarin buffer.

Ka'idar haɓaka matsuguni ta dogara ne akan gyare-gyaren sinadari na ƙuntatawa jerin tantancewar endonuclease a ƙarshen DNA ɗin da aka yi niyya.Ƙarshen endonuclease yana buɗe rata a cikin madaidaicin DNA a wurin gane shi, kuma DNA polymerase ya ƙaddamar da rata 3' Ƙarshen kuma ya maye gurbin DNA na gaba.

Za'a iya haɗa madauri guda ɗaya na DNA da aka maye gurbinsu tare da firamare kuma a faɗaɗa su zuwa nau'i biyu ta hanyar DNA polymerase.Ana maimaita wannan tsari akai-akai, ta yadda tsarin manufa ya inganta da kyau.

Abũbuwan amfãni da rashin amfanin fasahar haɓaka matsuguni

Amfanin SDA:

Ƙarfin haɓakawa yana da girma, lokacin amsawa yana da gajeren lokaci, ƙayyadaddun ƙayyadaddun yana da ƙarfi, kuma ba a buƙatar kayan aiki na musamman.

Nasarar SDA:

Kayayyakin ba iri ɗaya ba ne, kuma ana samar da wasu samfura masu ɗaiɗai ɗaya da mai ɗai-ɗai a koyaushe a cikin zagayowar SDA, kuma babu makawa wutsiya za ta faru idan electrophoresis ya gano.

Rolling da'irar ƙarawa

Rolling Circle amplification (RCA) an gabatar da shi ta hanyar zana hanyar yin kwafin DNA daga ƙwayoyin cuta ta hanyar birgima.Yana nufin yin amfani da DNA madauwari guda ɗaya a matsayin samfuri a madaidaicin zafin jiki, da kuma DNA polymerase na musamman (kamar Phi29) ) Ƙarƙashin aikin naɗaɗɗen da'irar DNA don cimma haɓakar kwayar halitta.

Ana iya raba RCA zuwa haɓakawa na linzamin kwamfuta da ƙarawa mai faɗi.Ingancin RCA na layi na iya kaiwa 10⁵sau, kuma ingancin madaidaicin RCA na iya kaiwa 10⁹sau.

Bambanci mai sauƙi, kamar yadda aka nuna a cikin hoton da ke ƙasa, haɓakawa na linzamin kwamfuta yana amfani da firamare 1 kawai, ƙarar ƙararrawa b yana da firamare 2.

RCA mai layi kuma ana kiranta RCA na farko.Maɗaukaki yana ɗaure ga DNA madauwari kuma yana haɓaka ta aikin DNA polymerase.Samfurin madaidaicin madauri ɗaya ne tare da adadi mai yawa na jerin maimaita sau dubbai tsawon madauki ɗaya.

Tun da samfurin RCA na layi yana haɗi koyaushe zuwa farkon farawa, sauƙin daidaita siginar shine babban fa'ida.

Exponential RCA, kuma aka sani da Hyper branched amplification HRCA (Hyper branched RCA), a cikin m RCA, daya firamare kara girman da RCA samfurin, na biyu firamare hybridizes tare da RCA samfurin da kuma mika, da kuma maye gurbin da aka riga an daure da RCA samfurin The downstream primers mika madauri, da kuma maimaita tsawo da kuma maye RCA a dendritic samfurin.

Fa'idodi da rashin amfanin mirginawar da'irar nucleic acid

Amfanin RCA:

Babban hankali, ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun abubuwa da sauƙin aiki.

Nasarar RCA:

Matsalolin bango yayin gano sigina.Yayin da RCA ke yi, binciken makullin makullin mara kewayawa da samfurin DNA ko RNA na binciken da ba a ɗaure ba na iya haifar da wasu sigina na bango. 

Nucleicacid na tushen haɓakawa

Nucleic acid-based amplification (NASBA) sabuwar fasaha ce da aka haɓaka bisa tushen PCR.Yana da ci gaba da haɓaka haɓakar acid nucleic acid wanda ke jagoranta ta hanyar ɓangarorin farko tare da jerin masu tallata T7.Fasaha na iya haɓaka samfurin RNA da kusan sau 109 a cikin kimanin awanni 2, wanda shine sau 1000 sama da tsarin PCR na al'ada kuma baya buƙatar kayan aiki na musamman.

Anyi amfani da wannan fasaha don saurin gano cututtuka da zarar ta bayyana, kuma kamfanoni da yawa a halin yanzu suna amfani da wannan hanyar a cikin kayan gano RNA.

Kodayake haɓakawa na RNA kuma na iya amfani da fasahar PCR ta juye-juye, NASBA tana da fa'idodinta: ana iya aiwatar da ita a ƙarƙashin yanayin zafi akai-akai, kuma ya fi kwanciyar hankali da daidaito fiye da fasahar PCR ta gargajiya.

Halin yana a ma'aunin Celsius 41 kuma yana buƙatar AMV (virus na myeloblastosis na Avian) mai jujjuya fassarar, RNase H, T7 RNA polymerase da nau'i-nau'i biyu don kammala.

Tsarin ya ƙunshi:

Fim ɗin gaba yana ƙunshe da jerin madaidaitan mai tallata T7.A lokacin amsawar, firam ɗin gaba yana ɗaure zuwa madaidaicin RNA kuma enzyme AMV ne ya ɗaure shi don samar da madauri biyu na DNA-RNA.

Rnase H yana narkewa RNA a cikin matasan sau biyu kuma yana riƙe da DNA guda ɗaya.

Ƙarƙashin aikin juyawa na baya da enzyme AMV, an kafa nau'i biyu na DNA wanda ke dauke da jerin masu gabatarwa na T7.

A ƙarƙashin aikin T7 RNA polymerase, an kammala aikin rubutun kuma an samar da adadi mai yawa na RNA mai niyya.

Amfanin NASBA:

(1) Mahimmancin sa yana da jerin masu talla na T7, amma DNA na kasashen waje guda biyu ba shi da jerin masu tallata T7 kuma ba za a iya ƙarawa ba, don haka wannan fasaha yana da ƙayyadaddun ƙayyadaddun bayanai da hankali.

(2) NASBA kai tsaye tana haɗa tsarin jujjuya juzu'i a cikin amsawar haɓakawa, rage lokacin amsawa.

Lalacewar NASBA:

(1) Abubuwan da ake ɗauka sun fi rikitarwa.

(2) Ana buƙatar nau'ikan enzymes iri uku don yin tsadar amsa.