I-PCR iwubuchwepheshe bokukhulisa i-nucleic acid obusetshenziswa kakhulu futhi busetshenziswa kakhulu ngenxa yokuzwela nokucaciswa kwayo.Kodwa-ke, i-PCR idinga ukushintshwa kwe-thermal okuphindaphindiwe futhi ayikwazi ukususa imikhawulo yokuthembela kumathuluzi nemishini, ekhawulela ukusetshenziswa kwayo ekuhlolweni kwenkundla yomtholampilo.

Kusukela ekuqaleni kwawo-1990, amalabhorethri amaningi aqale ukuthuthukisa ubuchwepheshe obuqhubekayo bokukhulisa izinga lokushisa obungadingi ukushintshwa kwe-thermal.Manje sebethuthukise ubuchwepheshe bokukhulisa i-loop-mediated isothermal isothermal, ubuchwepheshe bokukhulisa i-isothermal esikhundleni se-strand, ubuchwepheshe bokukhulisa umbuthano we-isothermal, kanye nokuncika kokulandelana kwe-nucleic acid.Ubuchwepheshe bokukhulisa i-Isothermal nobunye ubuchwepheshe. 

Li-oop-mediated isothermal amplification

Umgomo wokukhuliswa usekelwe eqinisweni lokuthi i-DNA isesimweni sokulingana esiguqukayo cishe ku-65°C.Uma noma iyiphi i-primer ibhangqwe ngesisekelo futhi yelulelwa engxenyeni ehambisanayo ye-DNA enemicu ekabili, omunye umucu uzohlukana futhi ube umucu owodwa.

Kuleli zinga lokushisa, i-DNA isebenzisa ama-primers aqondile angu-4 ukuthembela ku-DNA polymerase ye-strand-displacement ukwenza ukuhlanganiswa kwe-DNA-displacement DNA izijikeleze ngokuqhubekayo.

Okokuqala thola izifunda eziqondile ezingu-6 u-F3, F2, F1, B1, B2, B3 ohlotsheni lofuzo oluqondiwe, bese uklama ama-primer angu-4 ngokusekelwe kulezi zifunda eziqondile ezingu-6 (njengoba kuboniswe esithombeni esingezansi):

I-forward inner primer (FIP) yakhiwe i-F1c ne-F2.

I-backward inner primer (BIP) yakhiwe i-B1c ne-B2, kanti i-TTTT isetshenziswa njengesikhala phakathi nendawo.

Iziqalo zangaphandle ze-F3 kanye ne-B3 ngokulandelana kwazo zakhiwe izifunda ze-F3 ne-B3 ohlotsheni lofuzo oluqondiwe.

Kuhlelo lokusabela lwe-LAMP, ukugxila kwe-primer yangaphakathi izikhathi eziningana kune-primer yangaphandle.I-primer yangaphakathi iqala ihlanganiswe ne-template strand ukuze kuhlanganiswe umucu ohambisanayo ukuze kwakhiwe umucu ophindwe kabili we-DNA.Kamuva, i-primer yangaphandle ihlanganiswa ne-template strand ukuze kwakhiwe umucu ophindwe kabili we-DNA.Ngaphansi kwesenzo se-BstDNA polymerase, umucu ohambisanayo ohlanganiswe i-primer yangaphakathi uyakhululwa.Ngemva kochungechunge lokusabela, umucu ohambisanayo ekugcineni wakha umucu owodwa we-DNA onesakhiwo se-dumbbell.

I-dumbbell DNA umucu owodwa usetshenziswa njengesifanekiso ukuqhubeka sakha ukwakheka kwe-stem-loop ye-DNA enesiphetho esivulekile.Ama-primer angaphakathi nangaphandle aqondisa ukwakheka kwe-stem-loop ye-DNA ukuze kuqhubeke ukugudluka kwe-strand nokusabela kwesandiso, futhi ekugcineni kwakhiwe izakhiwo eziningi ze-stem-loop ezinobude obuhlukene.Ingxube ye-DNA.

Izinzuzo kanye nokubi kwe-loop-mediated isothermal amplification

Izinzuzo ze-LAMP:

(1) Ukusebenza kahle kokukhuliswa okuphezulu, okungakhulisa ngempumelelo amakhophi angu-1-10 wofuzo oluqondiwe phakathi kwehora elingu-1, futhi ukusebenza kahle kokukhulisa izikhathi eziyi-10-100 kune-PCR evamile.

(2) Isikhathi sokuphendula sifushane, ukucaciswa kunamandla, futhi akukho mishini ekhethekile edingekayo.

Ukushiyeka kwe-LAMP:

(1) Izidingo zama-primers ziphezulu kakhulu.

(2) Umkhiqizo okhulisiwe awukwazi ukusetshenziselwa ukwenza i-cloning nokulandelanisa, kodwa ungasetshenziswa kuphela ukwahlulela.

(3) Ngenxa yokuzwela kwayo okunamandla, kulula ukwenza ama-aerosols, okubangela amanga angamanga futhi kuthinte imiphumela yokuhlolwa.

Strand displacement amplification

I-Strand displacement amplification (SDA) iyindlela yokukhulisa i-DNA isothermal ye-in vitro esekelwe ekuphenduleni kwe-enzymatic okwahlongozwa okokuqala isazi saseMelika u-Walker ngo-1992.

Isistimu eyisisekelo ye-SDA ihlanganisa i-endonuclease evimbelayo, i-polymerase ye-DNA enomsebenzi wokususwa kwe-strand, amapheya amabili eziqalisi, ama-dNTP, nama-ion e-calcium ne-magnesium nezinhlelo zebhafa.

Umgomo wokukhuliswa kwe-strand displacement usekelwe ekulandeleni kokuqashelwa kwe-endonuclease okukhawulelwe ngamakhemikhali kuzo zombili iziphetho ze-DNA eqondiwe.I-endonuclease ivula igebe ku-DNA ye-strand endaweni yokuqashelwa kwayo, futhi i-DNA polymerase inweba igebe 3′ End futhi ithathe indawo ye-DNA strand elandelayo.

Imicu eyodwa eshintshiwe ye-DNA ingahlanganiswa neziqalo futhi inwetshwe ibe imicu ephindwe kabili nge-DNA polymerase.Le nqubo iphindaphindwa ngokuqhubekayo, ukuze ukulandelana okuhlosiwe kukhuliswe kahle.

Izinzuzo kanye nokubi kobuchwepheshe bokukhulisa i-strand displacement

Izinzuzo ze-SDA:

Ukusebenza kahle kwe-amplification kuphezulu, isikhathi sokuphendula sifushane, ukucaciswa kunamandla, futhi akukho mishini ekhethekile edingekayo.

Amaphutha e-SDA:

Imikhiqizo ayifani, futhi eminye imikhiqizo enemicu eyodwa nephindwe kabili ihlale ikhiqizwa emjikelezweni we-SDA, futhi umsila uzokwenzeka nakanjani lapho kutholwa i-electrophoresis.

Rolling circle amplification

I-Rolling circle amplification (RCA) ihlongozwa ngokudweba indlela yokukopisha i-DNA emagciwaneni amagciwane ngokuzungeza indilinga.Ibhekisela ekusetshenzisweni kwe-DNA eyindilinga ene-stranded eyodwa njengesifanekiso ekushiseni okungashintshi, kanye ne-DNA polymerase ekhethekile (njenge-Phi29) ) Ngaphansi kwesenzo sokugoqa ukuhlanganiswa kwe-DNA yesiyingi ukuze kuzuzwe ukukhuliswa kofuzo oluqondiwe.

I-RCA ingahlukaniswa ibe yi-linear amplification kanye ne-exponential amplification.Ukusebenza kahle kwe-RCA yomugqa kungafinyelela ku-10⁵izikhathi, kanye nokusebenza kahle kwe-RCA echazayo kungafinyelela ku-10⁹izikhathi.

Umehluko olula, njengoba kukhonjisiwe emfanekisweni ongezansi, ukukhulisa umugqa a kusebenzisa i-primer engu-1 kuphela, i-exponential amplification b ineziqalo ezi-2.

I-Linear RCA ibizwa nangokuthi i-single primer RCA.I-primer ibophezela ku-DNA eyindilinga futhi inwetshwa isenzo se-DNA polymerase.Umkhiqizo uwumugqa owodwa onenani elikhulu lokulandelana okuphindaphindwayo izikhathi eziyizinkulungwane ubude beluphu eyodwa.

Njengoba umkhiqizo we-RCA yomugqa uhlale uxhumeke ku-primer yokuqala, ukulungiswa okulula kwesignali kuyinzuzo enkulu.

I-Exponential RCA, eyaziwa nangokuthi i-Hyper branched amplification HRCA (Hyper branched RCA), ku-RCA exponential, i-primer eyodwa ikhulisa umkhiqizo we-RCA, i-primer yesibili ihlanganisa nomkhiqizo we-RCA futhi inwebeke, futhi esikhundleni sayo isivele iboshelwe kumkhiqizo we-RCA Iziqalo ezingezansi zinweba umucu, futhi ziphinda ukunwetshwa nokufaka esikhundleni somkhiqizo we-RCA amplification.

Izinzuzo kanye nokubi kwe-rolling circle nucleic acid amplification

Izinzuzo ze-RCA:

Ukuzwela okuphezulu, ukucaciswa okuhle nokusebenza okulula.

Amaphutha we-RCA:

Izinkinga zangemuva ngesikhathi sokutholwa kwesignali.Ngesikhathi sokusabela kwe-RCA, i-padlock probe engazuliswanga kanye nesifanekiso se-DNA noma i-RNA yophenyo olungaboshiwe kungase kukhiqize amasiginali athile angemuva. 

Ni-ucleicacid-based amplification esekelwe ngokulandelana

I-Nucleic acid sequence-based amplification (NASBA) ubuchwepheshe obusha obuthuthukiswe ngesisekelo se-PCR.Kuyi-amplification ye-nucleic acid eqhubekayo ne-isothermal eqondiswa ama-primers anokulandelana komgqugquzeli we-T7.Ubuchwepheshe bungakhulisa i-RNA yesifanekiso izikhathi ezingaba ngu-109 cishe emahoreni angu-2, ephakeme izikhathi ezingu-1000 kunendlela evamile ye-PCR futhi ayidingi imishini ekhethekile.

Lobu buchwepheshe busetshenziswe ekuxilongeni ngokushesha kwezifo ngokushesha nje lapho buvela, futhi izinkampani eziningi okwamanje zisebenzisa le ndlela kumakhithi okuthola i-RNA.

Nakuba ukukhulisa i-RNA kungase futhi kusebenzise ubuchwepheshe be-PCR bokubhalwe phansi, i-NASBA inezinzuzo zayo: ingenziwa ngaphansi kwezimo zokushisa ezilinganiselwe, futhi izinzile futhi inembe kakhulu kunobuchwepheshe be-PCR bendabuko.

Ukusabela kuku-41 degrees Celsius futhi kudinga i-AMV (avian myeloblastosis virus) reverse transcriptase, i-RNase H, i-T7 RNA polymerase kanye nepheya lezinto zokuqala ukuze kuqedwe.

Inqubo ikakhulukazi ihlanganisa:

I-primer eya phambili iqukethe ukulandelana okuhambisanayo komgqugquzeli we-T7.Ngesikhathi sokusabela, i-primer eya phambili ibophezela ku-RNA strand futhi icutshungulwa yi-enzyme ye-AMV ukuze yakhe i-DNA-RNA double strand.

I-RNase H igaya i-RNA ku-hybrid-strand kabili futhi igcina i-DNA enomucu owodwa.

Ngaphansi kwesenzo se-reverse primer kanye ne-enzyme ye-AMV, kwakheka umucu ophindwe kabili we-DNA oqukethe ukulandelana komgqugquzeli we-T7.

Ngaphansi kwesenzo se-T7 RNA polymerase, inqubo yokubhala iyaqedwa futhi inani elikhulu le-RNA eqondiwe liyakhiqizwa.

Izinzuzo ze-NASBA:

(1) I-primer yayo inokulandelana komgqugquzeli we-T7, kodwa i-DNA yangaphandle enemicu ephindwe kabili ayinakho ukulandelana komgqugquzeli we-T7 futhi ayikwazi ukukhuliswa, ngakho lobu buchwepheshe bunokucaciswa okuphezulu nokuzwela.

(2) I-NASBA ihlanganisa ngokuqondile inqubo yokuhlehla yokubhala ekuphenduleni kokukhulisa, ifinyeze isikhathi sokusabela.

Ukungalungi kwe-NASBA:

(1) Izingxenye zokusabela ziyinkimbinkimbi kakhulu.

(2) Kudingeka izinhlobo ezintathu zama-enzyme ukwenza ukusabela kubize kakhulu.