PCR, ndiyo polymerase chain reaction, iyo inoreva kuwedzera kwe dNTP, Mg2+, elongation zvinhu uye amplification ekusimudzira zvinhu kune sisitimu iri pasi pecatalysis yeDNA polymerase, uchishandisa mubereki DNA setemplate uye chaiyo primers senzvimbo yekutanga yekuwedzera. Kuburikidza nematanho e denaturation, annealing, extension, etc., maitiro e in vitro kudzokorora mwanasikana strand DNA inopindirana nemubereki strand template DNA inogona kukurumidza uye nekunyatso kusimudzira chero chinangwa cheDNA in vitro.
1. Kupisa Kutanga PCR
Nguva yekutanga yekuwedzera muPCR yakajairwa haisi yekuisa PCR muchina wePCR muchina, uye ipapo chirongwa chinotanga kuwedzera.Kana iyo sisitimu yekumisikidza yapera, kukwidziridzwa kunotanga, izvo zvinogona kukonzera kusiri-chaiyo amplification, uye inopisa-kutanga PCR inogona kugadzirisa dambudziko iri.
Chii chinopisa kutanga PCR?Mushure mekunge maitiro ekugadzirisa agadzirwa, iyo enzyme modifier inosunungurwa pakupisa kwepamusoro (kazhinji yakakwirira kudarika 90 ° C) panguva yekutanga kupisa kwekuita kana "hot start" nhanho, kuitira kuti DNA polymerase ishandiswe.Iyo chaiyo activation nguva uye tembiricha zvinoenderana nehunhu hweDNA polymerase uye inopisa-yekutanga modifier.Iyi nzira inonyanya kushandisa ma modifiers akadai sema antibodies, affinity ligands, kana makemikari modifiers kuvharira basa reDNA polymerase.Sezvo basa reDNA polymerase richidzivirirwa pakupisa kwekamuri, tekinoroji yekutanga inopisa inopa mukana wakakura wekugadzirira akawanda PCR maitiro masisitimu patembiricha yekamuri pasina kupira iyo chaiyo yePCR maitiro.
2. RT-PCR
RT-PCR (Reverse transcription PCR) inzira yekuyedza yekudzoreredza zvinyorwa kubva kumRNA kuenda kucDNA uye kuishandisa setemplate yekusimudzira.Maitiro ekuedza ndeekubvisa yakazara RNA mumatishu kana maseru kutanga, shandisa Oligo (dT) seprimer, shandisa reverse transcriptase kugadzira cDNA, uye wozoshandisa cDNA setemplate yePCR amplification kuti uwane iyo inotarirwa gene kana kuona gene expression.
3. Fluorescent quantitative PCR
Fluorescent quantitative PCR (Chaiyo-nguva Quantitative PCR,RT-qPCR) inoreva nzira yekuwedzera mapoka efluorescent kuPCR reaction system, uchishandisa kuunganidzwa kwezviratidzo zvefluorescent kutarisa yose PCR maitiro munguva chaiyo, uye pakupedzisira kushandisa chiyero chepamusoro chekuongorora template.Nzira dzinowanzo shandiswa qPCR dzinosanganisira SYBR Green I uye TaqMan.
4. Nested PCR
Nested PCR inoreva kushandiswa kwemaseti maviri ePCR maprimers maviri ePCR amplification, uye chigadzirwa chekusimudzira chechipiri chechipiri ndicho chinonangwa gene fragment.
Kana kusapindirana kwembiri yekutanga yemaprimers (outer primers) ikakonzera kuti chigadzirwa chisiri chakananga chikwidziridzwe, mukana weiyo dunhu rimwechete risiri chairo richizivikanwa nembiri yechipiri yeprimers uye kuramba uchikudza idiki kwazvo, saka iyo kukwidziridzwa neyechipiri peya yekutanga, iyo chaiyo PCR yakagadziridzwa.Imwe mukana wekuita marounds maviri ePCR ndeyekuti inobatsira kukwidziridza chigadzirwa chakaringana kubva kune mashoma kutanga DNA.
5. Bata pasi PCR
Touchdown PCR inzira yekuvandudza iyo chaiyo yePCR maitiro nekugadzirisa iyo PCR kutenderera paramita.
Mukubata-bata PCR, tembiricha yekumisikidza kwemashure mashoma ekutanga inogadzika madhigirii mashoma pamusoro peiyo tembiricha yekumisa (Tm) yemaprimers.Yepamusoro annealing tembiricha inogona kunyatso kudzikisa kusiri-chaiyo amplification, asi panguva imwe chete, yakakwirira annealing tembiricha inozowedzera kupatsanurwa kwemaprimers uye kutevedzana kwechinangwa, zvichikonzera kuderedzwa kwePCR goho.Naizvozvo, muzvikamu zvishoma zvekutanga, tembiricha yeannealing inowanzoiswa kuti iderere ne1 ° C per cycle kuti iwedzere zviri mukati meiyo gene gene muhurongwa.Kana tembiricha yekudziya yadzikiswa kusvika kune yakanyanya tembiricha, tembiricha yeannealing inochengeterwa kune asara mavhiri.
6. Yakananga PCR
Yakananga PCR inoreva kukwidziridzwa kwechinangwa cheDNA zvakananga kubva kumuenzaniso pasina kudiwa kweiyo nucleic acid yekuzviparadzanisa uye kucheneswa.
Kune marudzi maviri ePCR yakananga:
nzira yakananga: tora sampuli shoma uye woiwedzera zvakananga kuPCR Master Mix yekuzivikanwa kwePCR;
kuputika nzira: mushure mekuenzanisa sampuli, wedzera kune lysate, lyse kusunungura genome, tora zvishoma lysed supernatant uye uwedzere kuPCR Master Mix, ita PCR identification.Iyi nzira inorerutsa kufambisa kwekuedza, inoderedza maoko-nenguva, uye inodzivirira kurasikirwa kweDNA panguva yekucheneswa matanho.
7. SOE PCR
Gene splicing by overlap extension PCR (SOE PCR) inoshandisa maprimers ane magumo anopindirana kuita PCR zvigadzirwa zviumbe cheni dzinopindirana, kuitira kuti mukuzotevera amplification reaction, kuburikidza nekuwedzera kwemaketani anopokana, kwakasiyana masosi eA maitiro umo mazamu akakurisa anopindirana. uye zvakabatanidzwa pamwe chete.Iyi tekinoroji parizvino ine nzira mbiri dzekushandisa: kuvaka kwemajini efusion;gene site-directed mutation.
8. IPCR
Inverse PCR (IPCR) inoshandisa reverse complementary primers kukwidziridza zvimedu zveDNA kunze kwemaprimers maviri, uye inokudza kutevedzana kusingazivikanwe pamativi ese echimedu cheDNA chinozivikanwa.
IPCR yakatanga kugadzirwa kuti ione kutevedzana kwenzvimbo dziri pedyo dzisingazivikanwe, uye inonyanya kushandiswa kudzidza kutevedzana kwemajini ekusimudzira;oncogenic chromosomal rearrangements, zvakadai sejini fusion, translocation uye transposition;uye viral gene integration, inoshandiswawo inowanzoshandiswa ikozvino Kune site-directed mutagenesis, kopi plasmid neinoda mutation.
9. dPCR
Digital PCR (dPCR) inyanzvi yehuwandu hwehuwandu hwe nucleic acid mamorekuru.
Ikozvino kune nzira nhatu dzekuverengera nucleic acid mamorekuru.Photometry inobva pakunwa kwe nucleic acid molecules;real-time fluorescent quantitative PCR (Real Time PCR) inobva paCt value, uye Ct value inoreva nhamba yekutenderera inoenderana nehuwandu hwefluorescence hunogona kuonekwa;digital PCR ndiyo yazvino Quantitative tekinoroji yakavakirwa pane imwe-morekuru PCR nzira yekuverenga nucleic acid quantification ndiyo yakakwana yehuwandu nzira.
Nguva yekutumira: Jun-13-2023