PCR ndiye ukadaulo wogwiritsiridwa ntchito kwambiri wa nucleic acid amplification ndipo umagwiritsidwa ntchito kwambiri chifukwa cha chidwi chake komanso tsatanetsatane wake.Komabe, PCR imafuna kusinthasintha kwa kutentha kobwerezabwereza ndipo sikungathe kuchotsa malire odalira zida ndi zipangizo, zomwe zimalepheretsa kugwiritsidwa ntchito kwake pakuyesa kwachipatala.

Kuyambira koyambirira kwa zaka za m'ma 1990, ma laboratories ambiri ayamba kupanga ukadaulo wokulitsa kutentha womwe sufuna kutulutsa kutentha.Tsopano apanga ukadaulo wa loop-mediated isothermal amplification, ukadaulo wa strand m'malo mwa isothermal amplification, ukadaulo wozungulira wa isothermal amplification, komanso kudalira kwa nucleic acid.Tekinoloje ya Isothermal amplification ndi matekinoloje ena. 

Loop-mediated isothermal amplification

Mfundo yokulirapo imachokera pa mfundo yakuti DNA ili mumkhalidwe wosinthasintha wa pafupifupi 65 ° C.Chingwe chilichonse chikakhala chophatikizika ndikupititsidwa ku gawo lothandizira la DNA yamitundu iwiri, chingwe chinacho chimasiyanitsidwa ndikukhala chingwe chimodzi.

Pakutentha uku, DNA imagwiritsa ntchito zoyambira 4 kuti zidalira DNA polymerase ya strand-displacement kuti kaphatikizidwe ka strand-displacement DNA kudzizungulira mosalekeza.

Choyamba dziwani zigawo zisanu ndi chimodzi F3, F2, F1, B1, B2, B3 pa jini yomwe mukufuna, ndiyeno pangani zoyambira 4 kutengera zigawo 6 izi (monga momwe chithunzi chili pansipa):

The forward internal primer (FIP) imapangidwa ndi F1c ndi F2.

The backward inner primer (BIP) imapangidwa ndi B1c ndi B2, ndipo TTTT imagwiritsidwa ntchito ngati spacer pakati.

Zoyambira zakunja F3 ndi B3 motsatana zimapangidwa ndi zigawo za F3 ndi B3 pa jini yomwe mukufuna.

Mu njira ya LAMP reaction system, kuchuluka kwa primer yamkati kumakhala kangapo kuposa koyambira kunja.Choyambira chamkati chimayamba kuphatikizidwa ndi chingwe cha template kuti apange chingwe chothandizira kuti apange DNA iwiri.Pambuyo pake, choyambira chakunja chimaphatikizidwa ndi chingwe cha template kuti apange DNA iwiri chingwe.Pansi pa zochita za BstDNA polymerase, chingwe chothandizira chopangidwa ndi choyambirira chamkati chimatulutsidwa.Pambuyo pochita zinthu zingapo, chingwe chophatikizikacho chimapanga chingwe chimodzi cha DNA chokhala ndi dumbbell.

Dongosolo la dumbbell DNA single strand palokha imagwiritsidwa ntchito ngati template kuti ipitilize kupanga DNA yosinthira tsinde yokhala ndi malekezero otseguka.Zoyambira zamkati ndi zakunja zimatsogolera DNA yosinthira tsinde kuti isasunthike mosalekeza ndikuwonjezera, ndipo pamapeto pake imapanga ma tsinde angapo okhala ndi utali wosiyanasiyana.DNA mix.

Ubwino ndi kuipa kwa loop-mediated isothermal amplification

Ubwino wa LAMP:

(1) Kukweza kwakukulu, komwe kumatha kukulitsa makope a 1-10 amtundu womwe mukufuna mkati mwa 1h, ndipo mphamvu yokulitsa ndi 10-100 nthawi ya PCR wamba.

(2) Nthawi yochitira ndi yaifupi, kutsimikizika kwake ndikwamphamvu, ndipo palibe zida zapadera zomwe zimafunikira.

Zolakwika za LAMP:

(1) Zofunikira zoyambira ndizokwera kwambiri.

(2) Chowonjezeracho sichingagwiritsidwe ntchito popanga cloning ndi kutsatizana, koma chingagwiritsidwe ntchito pa chiweruzo.

(3) Chifukwa cha kukhudzidwa kwake kwakukulu, zimakhala zosavuta kupanga ma aerosols, zomwe zimapangitsa kuti zikhale zabodza komanso zimakhudza zotsatira za mayeso.

Skuwonjezereka kwa trand displacement

Strand displacement amplification (SDA) ndi in vitro isothermal DNA amplification njira yotengera enzymatic reaction yomwe idaperekedwa koyamba ndi katswiri waku America Walker mu 1992.

Dongosolo loyambira la SDA limaphatikizapo zoletsa endonuclease, DNA polymerase yokhala ndi strand displacement, ma pea awiri a primers, dNTPs, ndi calcium ndi magnesium ion ndi ma buffer systems.

Mfundo ya strand displacement amplification imachokera pa ndondomeko ya kuzindikira kwa endonuclease kumapeto kwa DNA yomwe mukufuna.Endonuclease imatsegula kusiyana kwa DNA ya chingwe pamalo ake odziwika, ndipo DNA polymerase imakulitsa kusiyana kwa 3 'Kumaliza ndikusintha chingwe chotsatira cha DNA.

Zingwe zosinthidwa za DNA zitha kuphatikizidwa ndi zoyambira ndikukulitsidwa kukhala zingwe ziwiri ndi DNA polymerase.Njirayi imabwerezedwa mosalekeza, kotero kuti ndondomeko ya chandamale imakulitsidwa bwino.

Ubwino ndi kuipa kwa ukadaulo wa strand displacement amplification

Ubwino wa SDA:

Kuchulukitsidwa kwachangu ndikokwera, nthawi yochitira ndi yaifupi, kutsimikizika kwake ndikwamphamvu, ndipo palibe zida zapadera zomwe zimafunikira.

Zolakwika za SDA:

Zogulitsa sizili zofananira, ndipo zinthu zina zokhala ndi chingwe chimodzi komanso zopindika ziwiri zimapangidwa nthawi zonse mumayendedwe a SDA, ndipo kutsetsereka kudzachitika mosapeŵeka kuzindikirika ndi electrophoresis.

Rkuchuluka kwa zozungulira

Rolling circle amplification (RCA) imaperekedwa pojambula njira yokopera DNA kuchokera ku tizilombo toyambitsa matenda mwa kuzungulira bwalo.Zimatanthawuza kugwiritsidwa ntchito kwa DNA yozungulira yozungulira imodzi monga template pa kutentha kosalekeza, ndi DNA polymerase yapadera (monga Phi29) ) Pansi pa kugudubuza kaphatikizidwe ka DNA kuti akwaniritse kukulitsa kwa jini yomwe mukufuna.

RCA ikhoza kugawidwa m'makulitsidwe amzere ndi kukulitsa kwachidziwitso.Kugwira ntchito kwa mzere wa RCA kumatha kufika 10⁵nthawi, ndi mphamvu ya RCA exponential akhoza kufika 10⁹nthawi.

Kusiyanitsa kosavuta, monga kuwonetseredwa m'chithunzi pansipa, kukulitsa mizere kumangogwiritsa ntchito 1 koyambirira, kukulitsa kwachidziwitso b kumakhala ndi zoyambira ziwiri.

Linear RCA imatchedwanso single primer RCA.Choyambirira chimamangiriza ku DNA yozungulira ndipo imakulitsidwa ndi zochita za DNA polymerase.Chogulitsacho ndi chingwe chimodzi chokhala ndi mizere yambiri yobwerezabwereza maulendo masauzande a kutalika kwa lupu limodzi.

Popeza kuti zopangidwa ndi RCA zofananira nthawi zonse zimagwirizanitsidwa ndi chiyambi choyambira, kukonza kosavuta kwa chizindikiro ndi mwayi waukulu.

Exponential RCA, yomwe imadziwikanso kuti Hyper branched amplification HRCA (Hyper branched RCA), mu exponential RCA, choyambirira chimodzi chimakulitsa mankhwala a RCA, choyambira chachiwiri chimasakanikirana ndi mankhwala a RCA ndikufalikira, ndipo m'malo mwake chimamangidwa kale ku mankhwala a RCA The zoyambira kumunsi zimakulitsa chingwe, ndikubwereza kukulitsa ndikusintha zinthu kuti apange RCA amplification.

Ubwino ndi kuipa kwa kugubuduza bwalo nucleic acid matalikidwe

Ubwino wa RCA:

Kuzindikira kwakukulu, kukhazikika bwino komanso ntchito yosavuta.

Zolakwika za RCA:

Mavuto am'mbuyo panthawi yozindikira chizindikiro.Pamachitidwe a RCA, kafukufuku wapadlock wosazunguliridwa ndi template ya DNA kapena RNA ya kafukufuku wosamangika atha kupanga zizindikiro zakumbuyo. 

Nucleicacid sequence-based amplification

Nucleic acid sequence-based amplification (NASBA) ndiukadaulo watsopano wopangidwa pamaziko a PCR.Ndiwopitilira komanso isothermal nucleic acid amplification motsogozedwa ndi zoyambira ziwiri zotsatizana ndi T7.Ukadaulo ukhoza kukulitsa template ya RNA ndi nthawi za 109 pafupifupi maola 2, omwe ndi nthawi 1000 kuposa njira ya PCR wamba ndipo safuna zida zapadera.

Tekinolojeyi yakhala ikugwiritsidwa ntchito pozindikira matenda mwachangu atangowonekera, ndipo makampani ambiri pakali pano amagwiritsa ntchito njirayi m'makina ozindikira a RNA.

Ngakhale kukulitsa kwa RNA kungagwiritsenso ntchito ukadaulo wa PCR wosinthira, NASBA ili ndi zabwino zake: imatha kuchitika pansi pa kutentha kosasintha, ndipo ndiyokhazikika komanso yolondola kuposa ukadaulo wamba wa PCR.

Zomwe zimachitika ndi madigiri 41 Celsius ndipo zimafunikira AMV (avian myeloblastosis virus) reverse transcriptase, RNase H, T7 RNA polymerase ndi ma primers kuti amalize.

Njirayi imaphatikizapo:

Choyambira chakutsogolo chimakhala ndi kutsatizana kotsatsira kwa T7.Panthawiyi, choyambira kutsogolo chimamangiriza ku chingwe cha RNA ndipo chimapangidwa ndi enzyme ya AMV kuti ipange DNA-RNA iwiri chingwe.

RNase H imagaya RNA mu hybrid double-strand ndikusunga DNA ya chingwe chimodzi.

Pansi pa zochita za reverse primer ndi enzyme ya AMV, chingwe cha DNA chawiri chokhala ndi mndandanda wa T7 chimapangidwa.

Pansi pa zochita za T7 RNA polymerase, njira yolembera imamalizidwa ndipo kuchuluka kwa chandamale cha RNA kumapangidwa.

Ubwino wa NASBA:

(1) Choyambirira chake chimakhala ndi mndandanda wa T7 wotsatsira, koma DNA yakunja yakunja ilibe ndondomeko ya T7 yotsatsira ndipo silingathe kukulitsidwa, kotero teknolojiyi imakhala ndi chidziwitso chapamwamba komanso chidziwitso.

(2) NASBA imaphatikizira mwachindunji njira yosinthira m'machitidwe amplification, kufupikitsa nthawi yochitira.

Zoyipa za NASBA:

(1) Zomwe zimapangidwira zimakhala zovuta kwambiri.

(2) Mitundu itatu ya michere imafunika kuti kachitidweko kakhale kokwera mtengo.