PCR, akawanda PCR, Iripo PCR, Reverse PCR, RT-PCR, qPCR (1)-PCR

Isu tichagadzirisa pfungwa, nhanho uye ruzivo rweakasiyana PCR

Ⅰ. PCR

Polymerase Chain Reaction, inodaidzwa kunzi PCR, tekinoroji yebhayoloji inoshandiswa kukudza zvimedu zveDNA.Inogona kutorwa seyakakosha DNA replication in vitro.DNA polymerase (DNA Polymerase I) yakawanikwa kare muna 1955, uye Klenow Fragment yeE. Coli, ine kukosha kwekuedza uye kushanda, yakawanikwa naDr. H. Klenow mukutanga kwema1970, asi nokuti iyi enzyme haibvumiri kupisa, kupisa kwepamusoro kunogona kuideredza, saka haisati yasangana nepolymerase degeneration chain reaction.Ma enzymes ari kushandiswa nhasi (anonzi Taq polymerase), akaparadzaniswa kubva kuThermus aquaticus, bhakitiriya rinopisa muchirimo muna 1976. Chimiro charo ndechokuti inogona kudzivisa kupisa kwepamusoro uye iine enzyme yakanaka, asi inoshandiswa zvakanyanya mushure memakore ekuma1980.Mafungiro epakutanga ekutanga echinyakare prototype yePCR yakafanana nekugadzirisa majini uye kukopa, izvo zvakakurudzirwa naDr. KJell Kleppe muna 1971. Akaburitsa yekutanga nyore uye pfupi-yenguva kopi yejeni (yakafanana neyakafanana nekutanga kutenderera kuviri kwePCR).PCR yakagadziridzwa nhasi yakagadzirwa naDr. Kary B. Mullis muna 1983. Dr. Mullis vakashandira makambani ePE gore iroro, saka PE ine chinzvimbo chakakosha muindasitiri yePCR.Dr. Mullis akabudisa zviri pamutemo pepa rekutanga rakabatana naSaiki nevamwe muna 1985. Kubva ipapo, kushandiswa kwePCR zviuru zvemakiromita pazuva, uye hutano hwemapepa ane hukama hunogona kunzi hunoita kuti dzimwe nzira dzakawanda dzekutsvakurudza dzive dzisina kunaka.Zvadaro, PCR tekinoroji inoshandiswa zvakanyanya mukutsvagisa biology yesainzi uye makiriniki mashandisirwo, ichiva iyo yakakosha tekinoroji yekutsvagisa mamorekuru biology.Mullis akahwinawo mubairo weNobel muna 1993 muChemistry.

PCRPrinciple

Nheyo yekutanga yePCR tekinoroji yakafanana neyechisikigo yekudzokorora maitiro eDNA, uye humbowo hwayo hunoenderana neiyo oligonucleotide primer inopindirana kumagumo ese ekutevedzana kwechinangwa.PCR inoumbwa nedegeneration-annealing-kuwedzera matanho matatu ekutanga ekuita: ①Degeneration of template DNA: Mushure mekunge template DNA yapisa kusvika 93°C kwenguva yakati rebei, iyo mbiri DNA mhinduro yehuviri-cheni DNA yakaumbwa nePCR kukwidziridzwa kwetemplate template DNA Kusiya, kuitira kuti igadzirire iyo imwe chete inotenderera.②Kubatanidzwa (musanganiswa) kwetemplate yeDNA uye yekutanga: Mushure mekunge template yeDNA yadziiswa uye yadzikiswa kuita cheni imwe chete, tembiricha inodonha kusvika pa55°C.Iyo inopindirana kutevedzana kweiyo primer uye template DNA imwe-ketani.③Kuwedzeredzwa kweiyo primer: DNA template-iyo yekutanga inosunga yakavakirwa pachiito cheTaqDNA polymerase, ine dNTP seyekuita mbishi zvinhu.Chengetedza musimboti wekudzokorodza, gadzira nyowani semi-yakachengeterwa kopi cheni inozadzisa iyo template yeDNA cheni, uye kudzokorora kutenderera kuderera-annealing-kuwedzera maitiro matatu anogona kuwana mamwe "semi-yakachengeterwa kopi cheni", uye iyi ketani itsva inowanikwa zvakare Iva template yeinotevera kutenderera.Zvinotora 2-4min kupedzisa loop, iyo inotarirwa gene inogona kukwidziridzwa mamirioni akati wandei mumaawa 2-3.

StandardPCRReaction System

Zvinhu zvishanu zvePCR reaction

Kune mhando shanu dzezvinhu zvinosanganisirwa muPCR kuita, zvinoti primer, enzyme, dNTP, template uye buffer (Mg2+ inodiwa).[PCR maitiro]

Iyo yakajairwa PCR maitiro akakamurwa kuita matanho matatu

1. DNA degeneration (90 ° C-96 ° C): Dual-chain DNA templates pasi pechiito chekupisa, hydrogen bonds break, ichiita DNA-chain chain.

2. Annealing (25 ℃ -65 ℃): Iyo tembiricha yehurongwa inoderedzwa, iyo primer inosanganiswa neDNA template kuti iite yemunharaunda mbiri-chain.

3. Kuwedzera (70 ℃ -75 ℃): Pasi pechiito cheTaq enzyme (inenge 72 ° C, basa rakanakisisa), dNTP inoshandiswa seyakasvibirira, inotambanudza kubva ku5′ kumagumo ekutanga → 3′ kuguma, synthesis uye template inopindirana neketani yeDNA.

Kutenderera kwega kwega kunodzorwa, kuvharirwa uye kuwedzerwa, kupeta kaviri zviri mukati meDNA.Parizvino, nekuda kwenzvimbo pfupi yekusimudzira, imwe PCR inogona kudzokororwa munguva pfupi kwazvo kunyange kana basa reTaq enzyme risina kunaka, saka rinogona kuchinjwa kuita matanho maviri, ndiko kuti, annealing uye kuwedzera kunogona kuitwa pa60 ° C-65 ° C panguva imwe chete.Kuti uderedze maitiro ekusimudza uye kutonhora uye kugadzirisa kukurumidza kwekupindura.

PCR Reaction maitiro

● Kunyatsotaura

Iwo chaiwo masikirwo emhinduro yePCR ndeaya: ①Musanganiswa chaiwo weprimer uye template yeDNA.②Nheyo yekubatanidza kwepasi.③Kuvimbika kweTaqDNA polymerase synthesis reaction.④Kujeka uye kuchengetedza kwejini rinotangwa.

Iko kusanganisa kwakaringana kwekutanga uye matemplate ndiyo kiyi.Iko kusungirirwa kweiyo primer uye template uye kuwedzera kweiyo primer chain kunoenderana nemusimboti we alkaline base matching.Kuvimbika kwepolymerase synthesis reactions uye yakakwirira tembiricha kuramba kweTaq DNA polymerase kugadzira iyo inosunga (musanganiswa) yetemplate uye primer mukuita inogona kuitwa pane yakanyanya tembiricha.Iyo chaiyo yekusanganiswa inowedzera zvakanyanya.Iyo clip inogona kuchengetedza yakakwira dhigirii yekururama.Nekusarudza nharaunda yakanangana neiyo genetic ine yakakwirira kuchengetedza uye yakakwirira kuchengetedza, iyo chaiyo yakakwira.

● Kunzwa Kunyanya

Nhamba yekugadzirwa kwePCR zvigadzirwa inowedzerwa ne index, iyo inogona kuwedzera template yekutanga yePicker (PG = 10-12) kuwedzera chiyero che microcontroller kusvika pamwero we micrograms (μg = -6).Masero anotarirwa anogona kuwonekwa kubva ku1 miriyoni maseru;mukuona mavhairasi, kunzwisiswa kwePCR kunogona kusvika 3 RFUs (isina mavara akaumbwa mayuniti);iyo shoma yekuona mwero mubhakitiriya sainzi mabhakitiriya matatu.

● Nyore uye Inokurumidza

Iyo PCR reflection inoshandisa high-temperature Taq DNA polymerase, iyo inowedzera mhinduro yekugadzirisa panguva imwe chete, kureva, kuora-anneal-extension reaction pane DNA amplification solution uye mvura yekugezera poto.Kazhinji, iyo amplification reaction inopedzwa mumaawa maviri kusvika mana.Zvigadzirwa zveAugmented zvinowanzoongororwa nebakatwa remagetsi, uye hazvifanirwe kushandisa isotopes, hapana kusvibiswa kweradioactive, uye kusimudzira kuri nyore.

● Kuchena kwemuenzaniso kwakaderera

Hapana chikonzero chekuparadzanisa mavhairasi kana mabhakitiriya uye masero etsika.DNA crude products uye RNA inogona kushandiswa seamplifiers.DNA amplification yekuona inogona kushandiswa zvakananga uchishandisa kiriniki zvienzaniso seropa, mvura yemuviri, mvura yekugeza kukosora, bvudzi, maseru, uye matishu mhenyu.

PCRmatambudziko akajairika

● Nhema dzenhema, hapana mabhendi akawedzerwa

Matanho akakosha ekuita kwePCR anosanganisira: ① kugadzirira kwetemplate nucleic acids, ② kunaka uye kujeka kwekutanga, ③ kunaka kwema enzymes ④ PCR kutenderera mamiriro.Kuwana chikonzero kunofanirawo kuongororwa uye kudzidzwa kune zviri pamusoro apa.

Matemplate: ① The template ine zvakasiyana-siyana mapuroteni, ② The template ine Taq enzyme inhibitor, ③ Protein iri mutemplate haina kubviswa, kunyanya puroteni yeboka muchromosome.⑤ Deminer nucleic acid degeneration haina kukwana.Kana mhando yema enzymes uye maprimers akanaka, hapana bhendi rekusimudzira, iro ringangove kurapa kwekugaya kwezvienzaniso.Pane chimwe chinhu chisina kunaka ne template nucleic acid extraction process, saka kugadzirira inoshanda uye yakagadzikana digestion mhinduro, maitiro ayo anofanira kugadziriswa uye kwete kuchinjwa.

Enzyme inactivation: enzyme itsva kana zvose zvekare uye zvitsva enzymes inofanira kushandiswa pamwe chete kuongorora kana basa re enzyme rakarasika kana kuti harina kukwana, zvichiita kuti zvive zvisina kunaka.Zvinofanira kucherechedzwa kuti Taq enzyme kana ethidium bromide dzimwe nguva inokanganwika.

Primer: mhando yeprimer, kusungirirwa kweiyo primer, uye kana iyo yekumisikidzwa kwemaprimers maviri ari symmetrical.Icho chikonzero chakajairika chekutadza kwePCR kana bhendi rinowedzera harina kunaka uye rinowanzopararira.Pane matambudziko nehunhu hwemaprimers edzimwe nhamba dzebatch.Iwo maviri maprimers ane yakanyanya kusungirirwa uye yakaderera yekumisikidza, zvichikonzera yakaderera-inoshanda asymmetric amplification.Iwo ekupikisa ndeaya: ① ​​Sarudza yakanaka primer yekugadzira mayunitsi.② Kusangana kweiyo primer hakungotsamira pane iyo OD kukosha, asi zvakare inoteerera kune yekutanga mvura yepakutanga kugadzira agar sugar gel electrophoresis.Panofanira kunge paine primer strip zone, uye kupenya kwemaprimers maviri kunofanirwa kuenderana.Bhandi, PCR inogona kutadza panguva ino, uye inofanirwa kugadziriswa neiyo primer synthesis unit.Kana primer yakakwira, kupenya kwakaderera, uye kusungirirwa kwayo kunofanirwa kuve kwakadzikama kana kwakaderedzwa.③ Iyo primer inofanirwa kubhadharwa uye kuchengetwa padanho repamusoro kudzivirira kutonhora kwakawanda kana kwenguva refu yefiriji zvikamu zvefiriji, izvo zvichaita kuti primer iparare uye iparare.④ Dhizaini yeprimer haina musoro, senge kureba kweiyo primer haina kukwana, uye di cluster inoumbwa pakati pekutanga.

Mg2 + concentration: Mg2 + ion concentration ine simba guru paPCR amplification kunyatsoshanda.Kunyanya kutarisisa kunogona kuderedza iyo yakasarudzika yePCR amplification.Kana iyo yekumisikidza yakadzikira, iyo PCR yekusimudzira inobuda inotoita iyo PCR yekusimudzira kutadza pasina bhendi yekuwedzera.

Shanduko yevhoriyamu yekuita: Vhoriyamu inoshandiswa muPCR kukwidziridzwa i20ul, 30ul, uye 50ul kana 100uL, iyo yakakura vhoriyamu yekushandisa kwePCR amplification inoiswa zvinoenderana nezvinangwa zvakasiyana zvekutsvagisa kwesainzi uye kuongororwa kwekiriniki.Mushure mekuita zvinyorwa zviduku zvakadai se20ul, zvinodikanwa kuita tambo mamiriro pakuita ukuru, kana zvisina kudaro ichakundikana.

Zvikonzero zvenyama: Shanduko yakakosha kune PCR kukwidziridzwa.Kana kupisa kwekushisa kwakaderera, nguva yekuderera ipfupi, zvinokwanisika kuitika mune zvakaipa zvenhema;yakaderera annealing tembiricha inogona kukonzera isiri-chaiyo amplification uye kuderedza chaiyo amplification kunyatsoita.Inobata zvakanyanya musanganiswa wemaprimers uye matemplate kudzikisa PCR amplification kunyatsoita.Dzimwe nguva zvinodikanwa kushandisa chiyero che thermometers kuona kusiyanisa, annealing uye yakawedzera tembiricha mukuwedzera kana mvura-soluble cooker, chinova chimwe chezvikonzero zvekutadza kwePCR.

Zvinangwa zvekutevedzana kwakasiyana: Kana iyo inoteedzana inoteedzana ikaitika, shanduko kana kudzima, musanganiswa weiyo prototype uye template yakasanganiswa, kana nekuda kwekushaikwa kwechinangwa chekutevedzana, iyo yekutanga uye template icharasikirwa inowirirana kutevedzana, uye PCR kukwidziridzwa kwayo hakuzobudiriri.

● Kunyepa

Iyo PCR amplification bhendi inoita seinoenderana neinotarirwa kutevedzana bhendi, uye dzimwe nguva bhendi rayo rinowedzera kutsvinda uye nepamusoro.

Primer dhizaini haina kukodzera: iyo yakasarudzwa yekumisikidza kutevedzana uye isiri-chinangwa amplification kutevedzana ine homologous, saka kana PCR inokwidziridzwa, iyo PCR yakasimudzwa zvigadzirwa hazvina-chinangwa kutevedzana.Kutevedzana kwechinangwa ipfupi kana kuti primer ipfupi, uye inowanzoitika kune yenhema positive.Inoda kugadzirwa patsva.

Kusvibiswa kwemuchinjiko wezvinangwa zvakatevedzana kana zvigadzirwa zvekusimudzira: Pane zvikonzero zviviri zvekusvibisa uku: Chekutanga, kuyambuka-kusvibiswa kwegenome yese kana zvikamu zvakakura, zvinotungamira kune zvenhema.Rudzi urwu rwekunyepa runogona kugadziriswa nenzira dzinotevera: Ngwarira uye unyoro panguva yekushanda kudzivirira kutevedzana kwechinangwa kubva mukupinzwa mupfuti yemuenzaniso kana kupfachuka kubva mucentrifugal chubhu.Kunze kwema enzymes uye zvinhu zvisingakwanise kumirisana nekupisa kwakanyanya, ese mareagents kana michina inofanirwa kuvharirwa hutachiona nekumanikidza kwakanyanya.Iyo centrifugal pombi uye sampuli dzinofanira kushandiswa panguva imwe chete.Kana zvichidikanwa, usati wawedzera zvienzaniso, iyo reaction chubhu uye reagent inoratidzirwa kune ultraviolet mwaranzi kuparadza iyo iripo nucleic acid.Chechipiri, zvimedu zviduku mukusvibiswa kwemhepo.Izvi zvimedu zvidiki zvipfupi pane zvakanangwa kutevedzana, asi zvine imwe homology.Inogona kusanganiswa pamwe chete.Mushure mekuzadzisa maprimers, chigadzirwa chePCR chinogona kuwedzerwa, izvo zvinozokonzera kugadzirwa kwakanaka kwenhema.Inogona kushandiswa kuderedza kana kubvisa dendere PCR nzira.

● Ratidza nonspecific amplification bhendi

Iwo mabhendi akaonekwa mushure mekukwidziridzwa kwePCR haaenderane nehukuru hunotarisirwa, kana hombe kana diki, kana panguva imwe chete, kana panguva imwe chete, mabhendi ekukudza uye asiri chaiwo mabhendi ekusimudza.Kubuda kwemabhendi asina-chaiyo ndeiyi: Chekutanga, maprimers haana kukwana anopindirana kune yakatarwa kutevedzana, kana polymerization yeprimer kuita di cluster.Chechipiri ndechekuti kusungirirwa kweMG2 + ion kwakakwira zvakanyanya, tembiricha yekudziya yakadzikira, uye huwandu hwePCR kutenderera hwakabatana.Chechipiri, kunaka uye huwandu hwema enzymes.Kazhinji, ma enzymes emamwe masosi anowanzo kune asiri-akakosha mabhendi uye ma enzymes eimwe sosi haana kuitika.Dzimwe nguva kwete-chaiyo amplification yema enzymes inoitikawo.Iwo anopikisa ndeaya: akagadziridzwa zvakare anokwezva kana zvichidikanwa.Deredza huwandu hwe enzyme kana kutsiva iyo enzyme yeimwe sosi.Deredza huwandu hwepuraimari, wedzera huwandu hwema templates nenzira kwayo, uye deredza huwandu hwekutenderera.Nyatsowedzera tembiricha yekuvharisa kana shandisa nzira mbiri dzetembiricha (93°C kudzikira, kuvharisa uye kuwedzera panenge pa65°C).

● Ratidza dhiri kana smear tepi

PCR amplification dzimwe nguva inoita seinoshandiswa kana kuvharwa kana kapeti-sebhandi.Nekuda kwechikonzero, nekuda kwehuwandu hwema enzymes kana hurombo husina kunaka hwee enzyme, iyo dNTP yekumisikidza yakanyanya, iyo Mg2 + yekumisikidza yakanyanya, tembiricha yekudziya yakadzikira, uye huwandu hwekutenderera hwakawandisa.Iwo ekupikisa ndeaya: ①Deredza huwandu hwema enzymes, kana shandura iyo enzyme yeimwe sosi.②Deretsa kuwanda kwe dNTP ③Kunyatso dzikisa Mg2+ kusungwa.④Wedzera huwandu hwematemplate uye kuderedza huwandu hwekutenderera.

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