PCR, zambiri PCR, Pa PCR, Kusintha kwa PCR, RT-PCR, qPCR (1)-PCR

Tidzakonza malingaliro, masitepe ndi tsatanetsatane wa PCR zosiyanasiyana

Ⅰ. PCR

Polymerase Chain Reaction, yomwe imatchedwa PCR, ndiukadaulo wachilengedwe wa mamolekyulu omwe amagwiritsidwa ntchito kukulitsa zidutswa za DNA.Itha kuwonedwa ngati kubwereza kwapadera kwa DNA mu vitro.DNA polymerase (DNA Polymerase I) inapezedwa kale mu 1955, ndipo Klenow Fragment ya E. Coli, yomwe ili ndi mtengo woyesera ndi zothandiza, inapezedwa ndi Dr. H. Klenow kumayambiriro kwa zaka za m'ma 1970, koma chifukwa chakuti puloteni iyi sichilola kutentha, kutentha kwakukulu kungathe kuipitsa, kotero sagwirizana ndi kutentha kwakukulu kwa polymerase chain reaction.Ma enzymes omwe amagwiritsidwa ntchito masiku ano (otchedwa Taq polymerase), adasiyanitsidwa ndi Thermus aquaticus, bakiteriya yotentha ya m'chaka cha 1976. Chikhalidwe chake ndi chakuti amatha kukana kutentha kwakukulu ndipo ndi enzyme yabwino, koma imagwiritsidwa ntchito kwambiri pambuyo pa zaka za m'ma 1980.Lingaliro lapachiyambi lachidziwitso choyambirira cha PCR ndi lofanana ndi kukonza jini ndi kukopera, zomwe zinaperekedwa ndi Dr. KJell Kleppe mu 1971.PCR yomwe idapangidwa lero idapangidwa ndi Dr. Kary B. Mullis ku 1983. Dr. Mullis adatumikira makampani a PE chaka chimenecho, kotero PE ili ndi udindo wapadera mumakampani a PCR.Dr. Mullis adafalitsa mwalamulo pepala loyamba logwirizana ndi Saiki ndi ena mu 1985. Kuyambira nthawi imeneyo, kugwiritsa ntchito PCR ndi zikwi za mailosi patsiku, ndipo ubwino wa mapepala okhudzana nawo ukhoza kunenedwa kuti umapangitsa njira zina zambiri zofufuzira kukhala zosasangalatsa.Pambuyo pake, ukadaulo wa PCR umagwiritsidwa ntchito kwambiri pakufufuza kwasayansi yazachilengedwe komanso kugwiritsa ntchito zamankhwala, kukhala ukadaulo wofunikira kwambiri pakufufuza kwa mamolekyulu a biology.Mullis adapambananso Mphotho ya Nobel mu Chemistry mu 1993.

PCRMfundo yofunika

Mfundo yayikulu yaukadaulo wa PCR ndi yofanana ndi njira yobwerezabwereza yachilengedwe ya DNA, ndipo kutsimikizika kwake kumadalira pa oligonucleotide primer yomwe imagwirizana ndi malekezero onse azomwe zatsata.PCR imapangidwa ndi kuchepa-kuwonjezera-kukulitsa njira zitatu zoyambira: ①Kuwonongeka kwa template ya DNA: template ya DNA ikatenthedwa kufika pafupifupi 93 ° C kwa nthawi inayake, njira yapawiri ya DNA yapawiri-chain DNA yopangidwa ndi PCR kukulitsa template ya template DNA Kusiya, kuti ipange chozungulira chotsatira kuti chigwirizane.②Kumangirira (compound) kwa template ya DNA ndi koyambira: DNA template ikatenthedwa ndi kusinthidwa kukhala tcheni chimodzi, kutentha kumatsika kufika pafupifupi 55°C.Kutsatira kophatikizana kwa choyambirira ndi template ya DNA single-chain.③Kukulitsidwa kwa choyambira: template ya DNA-kumangirira koyambira kumatengera zochita za TaqDNA polymerase, ndi dNTP ngati zopangira.Sungani mfundo yobwerezabwereza, phatikizani unyolo watsopano wosungika womwe umakwaniritsa unyolo wa DNA wa template, ndikubwereza njira zitatu zowonjezeretsa kubwerezabwereza, ndipo unyolo watsopanowu ukupezekanso Khalani template ya mkombero wotsatira.Zimatengera 2-4min kuti mutsirize kuzungulira, jini yachindunji ikhoza kukulitsidwa kangapo mamiliyoni ambiri mu maola 2-3.

StandardPCRReaction System

Zinthu zisanu za PCR reaction

Pali mitundu isanu yazinthu zomwe zimakhudzidwa ndi PCR, zomwe ndi primer, enzyme, dNTP, template ndi buffer (Mg2+ ndiyofunika).[Njira ya PCR]

Njira yokhazikika ya PCR imagawidwa m'magawo atatu

1. Kuwonongeka kwa DNA (90°C-96°C): Ma template a DNA a mitundu iwiri ya DNA pansi pa kutentha, hydrogen bond break, kupanga DNA ya unyolo umodzi.

2. Annealing (25 ℃ -65 ℃): Kutentha kwadongosolo kumachepetsedwa, choyambira chimaphatikizidwa ndi template ya DNA kuti ipange unyolo wapawiri.

3. Zowonjezera (70 ℃ -75 ℃): Pansi pa zochita za Taq enzyme (pafupifupi 72 ° C, ntchito yabwino kwambiri), dNTP imagwiritsidwa ntchito ngati zopangira, kufalikira kuchokera kumapeto kwa 5′ kumapeto kwa primer → 3′, kaphatikizidwe ndi template zimathandizirana wina ndi mnzake unyolo wa DNA.

Kuzungulira kulikonse kumasinthidwa, kulumikizidwa ndi kukulitsidwa, kuwirikiza kawiri zomwe zili mu DNA.Pakalipano, chifukwa cha malo afupikitsa amplification, PCR ina ikhoza kubwerezedwa mu nthawi yochepa kwambiri ngakhale ntchito ya Taq enzyme siili yabwino, kotero ikhoza kusinthidwa kukhala masitepe awiri, ndiko kuti, kuwonjezereka ndi kuwonjezereka kungathe kuchitidwa pa 60 ° C-65 ° C nthawi yomweyo.Pofuna kuchepetsa njira yokweza ndi kuziziritsa ndikuwongolera liwiro loyankha.

Zithunzi za PCR Reaction

● Mwachidule

Zomwe zimafunikira pakuyankhidwa kwa PCR ndi: ①Kuphatikizika kwapadera kwa choyambira ndi template ya DNA.② Mfundo yoyambira pawiri.③Kukhulupirika kwa TaqDNA polymerase synthesis reaction.④Kukhazikika ndi kusamala kwa jini yomwe mukufuna.

Kuphatikiza koyenera kwa zoyambira ndi ma templates ndiye chinsinsi.Kumanga kwa primer ndi template ndi kufalikira kwa unyolo woyambira kumatengera mfundo yofananira ndi alkaline base.Kukhulupirika kwa polymerase kaphatikizidwe kaphatikizidwe ndi kutentha kwapamwamba kwa Taq DNA polymerase kuti kumangiriza (compound) kwa template ndi primer mu zomwe zimachitika kutha kuchitidwa pa kutentha kwambiri.Chidziwitso cha kuphatikiza chikuwonjezeka kwambiri.The kopanira akhoza kukhala mkulu digiri yolondola.Posankha chigawo cha chibadwa chomwe chili ndi kusamala kwambiri komanso kusamala kwambiri, kutsimikizika kwake ndikwapamwamba.

● Kuzindikira Kwambiri

Kuchuluka kwa zinthu za PCR kumachulukitsidwa ndi index, yomwe imatha kukulitsa template yoyambira ya Picker (PG = 10-12) kuti iwonjezere mulingo wa microcontroller pamlingo wa micrograms (μg = -6).Maselo omwe amayang'aniridwa amatha kupezeka kuchokera ku maselo 1 miliyoni;pozindikira ma virus, kukhudzidwa kwa PCR kumatha kufikira 3 RFUs (malo opanda kanthu opangidwa mayunitsi);chiwerengero chochepa chodziwika mu sayansi ya bakiteriya ndi mabakiteriya atatu.

● Zosavuta komanso Zofulumira

Kuwonetsera kwa PCR kumagwiritsa ntchito kutentha kwapamwamba kwa Taq DNA polymerase, komwe kumawonjezera njira yothetsera nthawi imodzi, ndiko kuti, kuwonongeka kwa anneal-extension reaction pa DNA amplification solution ndi mphika wa madzi osamba.Nthawi zambiri, kukulitsa kumalizidwa mu 2 mpaka 4 maola.Zogulitsa zowonjezera nthawi zambiri zimawunikidwa ndi lupanga lamagetsi, ndipo sayenera kugwiritsa ntchito isotopu, kusaipitsa kwa radioactive, komanso kukwezedwa mosavuta.

● Kuyera kwa chitsanzo ndi kochepa

Palibe chifukwa cholekanitsa ma virus kapena mabakiteriya ndi ma cell achikhalidwe.DNA crude products ndi RNA zitha kugwiritsidwa ntchito ngati amplifiers.Kuzindikira kukulitsa kwa DNA kumatha kugwiritsidwa ntchito mwachindunji pogwiritsa ntchito zitsanzo zachipatala monga magazi, madzi amthupi, madzi ochapira chifuwa, tsitsi, maselo, ndi minofu yamoyo.

PCRmavuto wamba

● Zonama zabodza, zopanda ma amplified

Magawo ofunikira pakuchita kwa PCR ndi awa: ① Kukonzekera kwa ma nucleic acid a template, ② mtundu ndi tsatanetsatane wa zoyambira, ③ ma enzymes ④ PCR cycle cycle.Kupeza chifukwa kuyeneranso kuwunikiridwa ndikuphunziridwa pa maulalo omwe ali pamwambapa.

Ma templates: ① Tsambali lili ndi zomanga zosiyanasiyana, ② template ili ndi Taq enzyme inhibitor, ③ Mapuloteni omwe ali mu template samachotsedwa, makamaka mapuloteni agulu mu chromosome.⑤ Deminer nucleic acid degeneration sizokwanira.Ma enzymes ndi ma primers akakhala abwino, palibe gulu lokulitsa, lomwe ndi njira yochizira m'mimba ya zitsanzo.Pali cholakwika ndi template ya nucleic acid m'zigawo, kotero kukonzekera njira yabwino komanso yokhazikika ya chimbudzi, njira yake iyenera kukhazikitsidwa osati kusinthidwa mosasamala.

Kusagwira ntchito kwa enzyme: enzyme yatsopano kapena ma enzyme akale ndi atsopano ayenera kugwiritsidwa ntchito limodzi kuti awone ngati ntchito ya enzyme yatayika kapena yosakwanira, zomwe zimapangitsa kuti pakhale zolakwika zabodza.Tiyenera kudziwa kuti Taq enzyme kapena ethidium bromide nthawi zina amaiwala.

Choyambirira: mtundu wa zoyambira, kuchuluka kwa zoyambira, komanso ngati kuchuluka kwa zoyambira ziwirizo ndizofanana.Ndi chifukwa chofala cha kulephera kwa PCR kapena gulu lomwe likukulirakulira silabwino komanso losavuta kufalikira.Pali zovuta ndi mtundu wa zoyambira za manambala ena a batch.Zoyambira ziwirizi zimakhala ndi ndende yayikulu komanso yotsika kwambiri, zomwe zimapangitsa kuti asymmetric amplification ikhale yocheperako.Zotsutsana ndi izi: ① Sankhani choyambira chabwino kuti mupange mayunitsi.② Kuchuluka kwa primer sikungotengera mtengo wa OD, komanso kumayang'anira madzi oyambira kuti apange agar sugar gel electrophoresis.Payenera kukhala malo oyambira, ndipo kuwala kwa zoyambira ziwirizi kuyenera kukhala kofanana.Lamba, PCR ikhoza kulephera panthawiyi, ndipo iyenera kuthetsedwa ndi gawo loyambira.Ngati primer ndi yokwera, kuwala kwake kumakhala kochepa, ndipo kuyika kwake kuyenera kukhala koyenera pamene kuchepetsedwa.③ The primer iyenera kulipidwa ndikusungidwa pamalo okwera kwambiri kuti mupewe kuzizira kambiri kapena kuzizira kwanthawi yayitali mufiriji, zomwe zingapangitse kuti choyambiriracho chiwonongeke ndikuwonongeka.④ Mapangidwe a primer ndi osamveka, monga kutalika kwa primer sikukwanira, ndipo di cluster imapangidwa pakati pa zoyambira.

Kukhazikika kwa Mg2 +: Kukhazikika kwa Mg2 +ion kumakhudza kwambiri kukulitsa kwa PCR.Kuyika kwambiri kumatha kuchepetsa kugonana kosiyana ndi kukula kwa PCR.Ngati ndendeyo ili yotsika kwambiri, kutulutsa kwa PCR kumapangitsa kuti PCR iwonongeke popanda gulu lokulitsa.

Kusintha kwa voliyumu yamachitidwe: Voliyumu yomwe imagwiritsidwa ntchito pakukulitsa kwa PCR ndi 20ul, 30ul, ndi 50ul kapena 100uL, voliyumu yayikulu yogwiritsira ntchito kukulitsa kwa PCR imayikidwa molingana ndi zolinga zosiyanasiyana za kafukufuku wasayansi ndi kuyezetsa kwachipatala.Pambuyo popanga mavoti ang'onoang'ono monga 20ul, m'pofunika kupanga chikhalidwe cha chingwe popanga kukula, mwinamwake chidzalephera.

Zifukwa zakuthupi: Kusintha ndikofunikira kwambiri pakukulitsa kwa PCR.Ngati kutentha kwapang'onopang'ono kuli kochepa, nthawi yowonongeka ndi yochepa, zikhoza kuchitika mu zolakwika zabodza;Kutentha kwambiri kwa annealing kungayambitse kukweza kosadziwika bwino ndikuchepetsa magwiridwe antchito enieni.Zimakhudza kwambiri kuphatikiza kwa zoyambira ndi ma templates kuti muchepetse kukulitsa kwa PCR.Nthawi zina m`pofunika ntchito muyezo thermometers kuti azindikire kusinthasintha, annealing ndi yaitali kutentha mu kutambasuka kapena madzi -soluble cooker, chomwe ndi chimodzi mwa zifukwa kulephera kwa PCR.

Zosintha zotsatizana za chandamale: Ngati ndondomeko ya chandamale ichitika, kusintha kapena kuchotsedwa, kuphatikiza kwa pulojekiti ndi template kuphatikizidwa, kapena chifukwa cha kusowa kwa ndondomeko ya chandamale, choyambira ndi template zidzataya ndondomeko yowonjezera, ndipo kukulitsa kwa PCR sikungapambane.

● Zonama

Gulu lokulitsa la PCR limawoneka logwirizana ndi gulu lotsatizana, ndipo nthawi zina gulu lake limakhala laudongo komanso lalitali.

Kupanga koyambira sikoyenera: kutsatizana kokulira kosankhidwa ndi kukwezera kopanda cholinga kumakhala kofanana, kotero pamene kukulitsa kwa PCR, zinthu za PCR zokulirapo sizikhala ndi cholinga.Kutsata kwazomwe mukufuna kutsata ndi kwakufupi kwambiri kapena koyambira ndi kwakufupi kwambiri, ndipo kumakonda kukhala zabodza.Kufunika kukonzedwanso.

Kuipitsidwa kwapang'onopang'ono kwa zinthu zotsatizana kapena zokulitsa: Pali zifukwa ziwiri zakuipitsa izi: Choyamba, kuipitsidwa kwamtundu wonse kapena zigawo zazikulu, zomwe zimatsogolera ku zabwino zabodza.Mtundu wabodza wamtunduwu utha kuthetsedwa ndi njira izi: Samalani komanso mofatsa mukamagwira ntchito kuti mupewe kutengera zomwe mukufuna kuti zisalowe mumfuti yachitsanzo kapena kuwomba kuchokera mu chubu chapakati.Kupatula ma enzyme ndi zinthu zomwe sizingathe kupirira kutentha kwakukulu, ma reagents kapena zida zonse ziyenera kutetezedwa ndi tizilombo toyambitsa matenda.Mipope ya centrifugal ndi zitsanzo ziyenera kugwiritsidwa ntchito nthawi imodzi.Ngati ndi kotheka, pamaso kuwonjezera toyesa, zimene chubu ndi reagent ndi poyera ndi cheza ultraviolet kuwononga alipo nucleic asidi.Chachiwiri, tiziduswa tating'onoting'ono mu kuipitsidwa kwa mpweya.Tizidutswa tating'onoting'ono timeneti ndi zazifupi kuposa momwe chandamale, koma zili ndi ma homology ena.Ikhoza kuphatikizidwa ndi wina ndi mzake.Pambuyo powonjezera zoyambira, chinthu cha PCR chikhoza kukulitsidwa, zomwe zingayambitse kupanga zabwino zabodza.Itha kugwiritsidwa ntchito kuchepetsa kapena kuthetsa njira ya PCR ya chisa.

● Mawonekedwe a bandi yokwezeka yosatchulidwe

Magulu omwe adawonekera pambuyo pakukulitsa kwa PCR ndi osagwirizana ndi kukula koyembekezeredwa, kapena zazikulu kapena zazing'ono, kapena nthawi yomweyo, kapena nthawi imodzimodziyo, magulu apadera okulitsa ndi magulu osakhazikika.Kutuluka kwa magulu osakhala achindunji ndi: Choyamba, zoyambira ndizosakwanira kutsatana ndi zomwe mukufuna, kapena polymerization ya choyambira kupanga di cluster.Chachiwiri ndikuti kuchuluka kwa ma MG2 + ion ndikokwera kwambiri, kutentha kwa annealing ndikotsika kwambiri, ndipo kuchuluka kwa ma PCR kumalumikizidwa.Kachiwiri, ubwino ndi kuchuluka kwa michere.Nthawi zambiri, ma enzymes ochokera kuzinthu zina amakhala osagwirizana ndi magulu omwe si apadera ndipo ma enzymes amtundu wina samachitika.Nthawi zina, ma enzymes amakulirakulira mwanjira inayake.Zotsutsana nazo ndi: kukonzanso zokopa ngati kuli kofunikira.Chepetsani kuchuluka kwa enzyme kapena kusintha enzyme ya gwero lina.Chepetsani kuchuluka kwa zoyambira, onjezerani kuchuluka kwa ma template moyenera, ndikuchepetsa kuchuluka kwa mizere.Wonjezerani bwino kutentha kwa annealing kapena gwiritsani ntchito njira ziwiri za kutentha (93 ° C degeneration, annealing and extending at about 65°C).

● Mawonekedwe a ng'anjo yosalala kapena tepi yopaka

Kukulitsa kwa PCR nthawi zina kumawoneka ngati kumayikidwa kapena kutsekedwa kapena lamba ngati lamba.Pazifukwa zake, chifukwa cha kuchuluka kwa michere kapena kusakwanira kwa enzyme, kuchuluka kwa dNTP ndikokwera kwambiri, kuchuluka kwa Mg2 + ndikokwera kwambiri, kutentha kwa annealing ndikotsika kwambiri, ndipo kuchuluka kwa mikombero kumakhala kochulukirapo.Njira zothana nazo ndi izi: ①Chepetsani kuchuluka kwa ma enzymes, kapena sinthani ma enzyme a gwero lina.②Chepetsani kuchuluka kwa dNTP ③Chepetsani bwino kuchuluka kwa Mg2+.④Onjezani kuchuluka kwa ma tempuleti ndikuchepetsa kuchuluka kwa mikombero.

Zogwirizana nazo

PCR Heroᵀᴹ (Yokhala Ndi Dye)

◮ Kukhulupirika kwakukulu: kuwirikiza ka 6 kuposa enzyme wamba ya Taq;

◮ Liwiro lokulitsa kwambiri

◮ Kusintha kwa ma template ambiri

◮ Kukweza kwakukulu

◮ Kulekerera kwa chilengedwe kuli kolimba: kuikidwa pa 37 ° C kwa sabata, kusunga ntchito zoposa 90%;

◮ Ili ndi 5'→3' DNA polymerase zochita ndi 5'→3' exonuclease zochita, popanda 3'→5' ntchito exonuclease.

PCR Easyᵀᴹ (Yokhala Ndi Dye)

Dongosolo lapadera la machitidwe komanso magwiridwe antchito apamwamba a Taq DNA Polymerase amapangitsa kuti ma PCR azichita bwino kwambiri, amvekere komanso amvekere.

RT-qPCR Easyᵀᴹ (Chigawo Chimodzi) -SYBR Green I

◮ Kiti ya sitepe imodzi imapanga zolembera mobwereza ndi qPCR machitidwe awiri mu chubu chimodzi, zimangofunika kuwonjezera RNA yachifanizo, zoyambira za PCR ndi RNase-Free ddH₂O.

◮ Zida zimatha kusanthula mwachangu komanso moyenera ma virus a RNA kapena kufufuza RNA.

◮ Zidazi zimagwiritsa ntchito makina apadera a Foregene reverse transcription reagent ndi Foregene HotStar Taq DNA Polymerase ophatikizidwa ndi njira yapadera yochitira zinthu kuti apititse patsogolo kukulitsa bwino komanso momwe zimachitikira.

◮ Dongosolo lokonzekera bwino lomwe limapangitsa kuti zomwe zimachitikazo zikhale ndi chidwi chodziwikiratu, kukhazikika kwamafuta, komanso kulolerana bwino.

◮ RT-qPCR Yosavuta^TM(One Step) -SYBR Green I kit imabwera ndi utoto wamkati wa ROX, womwe ungagwiritsidwe ntchito kuthetsa zolakwika zakumbuyo ndi ma sign pakati pa zitsime, zomwe ndizosavuta kuti makasitomala azigwiritsa ntchito mitundu yosiyanasiyana ya zida za PCR.

RT Easy^TMII (Master Premix for woyamba chingwe cDNA kaphatikizidwe kwaReal Time PCR)

-Kutha kuchotsa gDNA, yomwe imatha kuchotsa gDNA mu template mkati mwa mphindi ziwiri.

-Njira yabwino yolembera, zimangotenga mphindi 15 kuti amalize kaphatikizidwe ka chingwe choyamba cha cDNA.

-Ma tempulo ovuta: ma tempulo okhala ndi GC yayikulu komanso mawonekedwe achiwiri ovuta amathanso kusinthidwa ndikuchita bwino kwambiri.

-High-sensitivity reverse transcript system, ma tempulo a pg-level amathanso kupeza cDNA yapamwamba kwambiri.

-Makina osindikizira a reverse ali ndi kukhazikika kwa kutentha kwambiri, kutentha kwabwino kwambiri ndi 42 ℃, ndipo akadali ndi ntchito yabwino yosinthira ku 50 ℃.