PCR ndiyo inonyanya kushandiswa nucleic acid amplification tekinoroji uye inoshandiswa zvakanyanya nekuda kwekunzwa kwayo uye kujeka.Nekudaro, PCR inoda kudzokororwa kupisa denaturation uye haigone kubvisa zvipingamupinyi zvekuvimba nezviridzwa uye zvishandiso, izvo zvinomisa kushanda kwayo mukiriniki yekuongorora ndima.

Kubva kutanga kwema1990s, marabhoritari mazhinji akatanga kugadzira tekinoroji yekusimudzira tembiricha isingade kupisa kwekupisa.Ikozvino ivo vakagadzira loop-mediated isothermal amplification tekinoroji, strand inotsiva isothermal amplification tekinoroji, rolling denderedzwa isothermal amplification tekinoroji, uye nucleic acid sequence kutsamira.Isothermal amplification tekinoroji uye mamwe matekinoroji. 

Loop-mediated isothermal amplification

The amplification musimboti wakavakirwa pakuti DNA iri mu dynamic equilibrium state at about 65°C.Kana chero primer iri base-paired uye yakatambanudzirwa kune inopindirana chikamu chekaviri-tambo DNA, imwe tambo inoparadzana uye ive imwechete-tambo.

Pane tembiricha iyi, DNA inoshandisa 4 chaiwo maprimers kuvimba nestrand-displacement DNA polymerase kuita synthesis yestrand-displacement DNA inotenderera ichienderera.

Chekutanga tarisa matunhu matanhatu F3, F2, F1, B1, B2, B3 pajena rinotariswa, wozogadzira 4 maprimers zvichienderana nematunhu matanhatu aya (sezvinoratidzwa pamufananidzo uri pazasi):

Iyo yekumberi yemukati primer (FIP) inoumbwa neF1c uye F2.

Iyo yekumashure yemukati primer (BIP) inoumbwa neB1c neB2, uye TTTT inoshandiswa se spacer pakati.

Iwo ekunze maprimers F3 neB3 akateedzana anoumbwa neF3 neB3 matunhu pane inotangwa gene.

Mune iyo LAMP reaction system, kusungirirwa kwemukati primer kwakapetwa kakati wandei kune yekunze kwekutanga.Iyo yemukati primer inotanga kusanganiswa ne template strand kuti igadzire tambo inopindirana kugadzira DNA kaviri tambo.Zvadaro, iyo yekutanga primer inosanganiswa ne template strand kuti iite DNA kaviri tambo.Pasi pechiito cheBstDNA polymerase, iyo inopindirana tambo yakasanganiswa neyemukati primer inoburitswa.Mushure mekuita kwakatevedzana, tambo inopindirana inozogadzira tambo imwe yeDNA ine dumbbell chimiro.

Iyo dumbbell chimiro DNA single strand pachayo inoshandiswa se template kuti irambe ichigadzira inoshandura stem-loop chimiro DNA ine yakavhurika magumo.Iwo emukati uye ekunze maprimers anotungamira inoshandura stem-loop chimiro DNA kuti irambe ichienda kune strand displacement uye ekuwedzera maitiro, uye pakupedzisira inogadzira akawanda stem-loop zvimiro zvine hurefu hwakasiyana.DNA musanganiswa.

Zvakanakira uye zvakaipira zve loop-mediated isothermal amplification

Zvakanakira zveLAMP:

(1) Kukwirisa kwepamusoro kunyatsoita, iyo inogona kunyatso kusimudzira 1-10 makopi einotariswa gene mukati me1h, uye kugona kwekusimudzira ndeye 10-100 nguva yeyakajairika PCR.

(2) Iyo nguva yekuita ipfupi, iyo chaiyo yakasimba, uye hapana yakakosha midziyo inodiwa.

Kukanganisa kweLAMP:

(1) Zvinodiwa zvemaprimers zvakanyanya kukwirira.

(2) Chigadzirwa chakasimudzirwa hachigone kushandiswa kugadzira uye kutevedzana, asi chinogona kungoshandiswa kutonga.

(3) Nekuda kwekunzwa kwayo kwakasimba, zviri nyore kugadzira aerosols, zvichikonzera manyepo enhema uye kukanganisa mhinduro dzebvunzo.

Strand displacement amplification

Strand displacement amplification (SDA) is in vitro isothermal DNA amplification nzira yakavakirwa pane enzymatic reaction yakatanga kutaurwa neAmerican nyanzvi Walker muna 1992.

Iyo yakakosha sisitimu yeSDA inosanganisira inorambidza endonuclease, DNA polymerase ine strand displacement chiitiko, maviri maviri ekutanga, dNTPs, uye calcium uye magnesium ion uye buffer masisitimu.

Iyo musimboti we strand displacement amplification yakavakirwa pane kemikari yakagadziriswa restriction endonuclease recognition sequence kumativi ese ari maviri echinangwa cheDNA.Iyo endonuclease inovhura gap mu strand DNA panzvimbo yayo yekuzivikanwa, uye DNA polymerase inowedzera gap 3 'Kupera uye kutsiva inotevera DNA strand.

Iyo yakatsiviwa tambo imwe chete yeDNA inogona kusanganiswa nemaprimers uye kuwedzerwa kuita tambo mbiri neDNA polymerase.Iyi nzira inodzokororwa nguva dzose, kuitira kuti kutevedzana kwechinangwa kuwedzere.

Zvakanakira uye zvakaipira zve strand displacement amplification tekinoroji

Zvakanakira zveSDA:

Kushanda kweamplification kwakakwira, nguva yekuita ipfupi, iyo chaiyo yakasimba, uye hapana yakakosha michina inodiwa.

Kukanganisa kweSDA:

Zvigadzirwa hazvina yunifomu, uye zvimwe zvigadzirwa zvine tambo imwe chete uye kaviri-tambo zvinogara zvichigadzirwa muSDA kutenderera, uye miswe inozoitika kana yaonekwa ne electrophoresis.

Rolling denderedzwa amplification

Rolling denderedzwa amplification (RCA) inokurudzirwa nekudhirowa panzira yekukopa DNA kubva kune pathogenic organisms nekutenderedza denderedzwa.Inoreva kushandiswa kwekamwe-stranded denderedzwa DNA se template pane inogara tembiricha, uye yakakosha DNA polymerase (yakadai sePhi29) ) Pasi pechiito chekutenderedza denderedzwa DNA synthesis kuti iwane kukwidziridzwa kwechinangwa gene.

RCA inogona kukamurwa kuita mutsara amplification uye exponential amplification.Kushanda kwemutsara RCA kunogona kusvika gumi⁵nguva, uye kushanda kweexponential RCA kunogona kusvika gumi⁹nguva.

Musiyano wakapfava, sezvinoratidzwa pamufananidzo uri pazasi, mutsara wekusimudzira a inoshandisa 1 primer, exponential amplification b ine 2 maprimers.

Linear RCA inonziwo single primer RCA.A primer inosunga denderedzwa DNA uye inowedzerwa nekuita kweDNA polymerase.Chigadzirwa chacho chine mutsara tambo imwe ine nhamba huru yekudzokorora kutevedzana zviuru zvenguva kureba kwechishwe chimwe chete.

Sezvo chigadzirwa chemutsara RCA chinogara chakabatana kune yekutanga primer, kugadzirisa nyore kwechiratidzo chinhu chikuru chakanakira.

Exponential RCA, inozivikanwawo seHyper branched amplification HRCA (Hyper branched RCA), muexponential RCA, imwe primer inokudza chigadzirwa cheRCA, yechipiri primer inosanganiswa neRCA chigadzirwa uye inowedzera, uye inotsiva yatosungirirwa kune chigadzirwa cheRCA Iwo epasi pemvura anowedzera tambo, uye anodzokorora kuwedzera uye kutsiva kugadzira RCA denderedzwa.

Zvakanakira uye kuipa kwekutenderedza denderedzwa nucleic acid amplification

Zvakanakira zveRCA:

High senitivity, yakanaka chaiyo uye nyore kushanda.

Kukanganisa kweRCA:

Matambudziko ekumashure panguva yekuona chiratidzo.Munguva yekuitwa kweRCA, iyo isina kutenderedzwa padlock probe uye template DNA kana RNA yeiyo isina kusungwa probe inogona kuburitsa mamwe masaini masaini. 

Nucleicacid sequence-based amplification

Nucleic acid sequence-based amplification (NASBA) tekinoroji nyowani yakagadzirwa pahwaro hwePCR.Iyo inoenderera uye isothermal nucleic acid amplification inotungamirwa nepeya yekutanga ine T7 inosimudzira kutevedzana.Iyo tekinoroji inogona kukudza iyo template RNA nekanosvika 109 mumaawa maviri, inova yakakwira ka1000 pane yakajairika PCR nzira uye haidi yakakosha michina.

Iyi tekinoroji yakashandiswa kukurumidza kuongororwa kwezvirwere payakangoonekwa, uye makambani mazhinji parizvino anoshandisa nzira iyi muRNA yekuona kits.

Kunyangwe RNA amplification inogona zvakare kushandisa reverse transcription PCR tekinoroji, NASBA ine zvayakanakira: inogona kuitwa pasi pemamiriro ekushisa anogara aripo, uye yakagadzikana uye yakarurama kupfuura tekinoroji yePCR.

Mhedzisiro yacho iri pa41 degrees Celsius uye inoda AMV (avian myeloblastosis virus) reverse transcriptase, RNase H, T7 RNA polymerase uye peya yemaprimers kuti ipedze.

Iyo nzira inonyanya kusanganisira:

Iyo yekumberi primer ine inopindirana kutevedzana kweT7 anosimudzira.Munguva yekuita, iyo yekutanga primer inosunga kuRNA strand uye inogadziriswa neAMV enzyme kuti igadzire DNA-RNA kaviri tambo.

RNase H inogaya RNA muhybrid kaviri-strand uye inochengeta DNA ine tambo imwe chete.

Pasi pechiito che reverse primer uye AMV enzyme, DNA kaviri tambo ine T7 inosimudzira kutevedzana inoumbwa.

Pasi pechiito cheT7 RNA polymerase, maitiro ekunyora anopedzwa uye huwandu hukuru hwechinangwa RNA hunogadzirwa.

Zvakanakira zveNASBA:

(1) Yayo yekutanga ine T7 inosimudzira kutevedzana, asi yekunze-yakasungwa kaviri DNA haina T7 inosimudzira kutevedzana uye haigone kukwidziridzwa, saka tekinoroji iyi ine hunyanzvi hwepamusoro uye kunzwa.

(2) NASBA inobatanidza zvakananga reverse transcription process muamplification reaction, ichipfupisa nguva yekuita.

Zvakaipa zveNASBA:

(1) Maitiro ekuita akanyanya kuoma.

(2) Matatu marudzi emaenzayimu anodiwa kuita kuti mhinduro iwedzere.