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Tekinoroji yekuongorora tekinoroji inoshandisa ma molecular biology nzira kuona kutaura uye chimiro cheiyo genetic zvinhu zvemuviri wemunhu uye hutachiona hwakasiyana, kuitira kuti uwane chinangwa chekufanotaura uye kuongorora zvirwere.

Mumakore achangopfuura, nekuvandudzwa uye kudzokorora tekinoroji yekuongorora mamorekuru, iko kushandiswa kwekiriniki yekuongorora kwema molecular kwawedzera kuwanda uye kwakadzama, uye musika wekuongorora mamorekuru wapinda munguva yekukurumidza kukura.

Munyori anopfupikisa akajairwa mamorekuru ekuongorora matekinoroji pamusika, uye akakamurwa kuita zvikamu zvitatu: chikamu chekutanga chinozivisa tekinoroji yePCR, chikamu chechipiri chinosuma iyo nucleic acid isothermal amplification tekinoroji, uye chikamu chechipiri chinosuma tekinoroji yekutevedzana.

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Chikamu I: PCR Technology

PCR tekinoroji

PCR (polymerase chain reaction) ndeimwe yein vitro DNA amplification technologies, ine nhoroondo yemakore anopfuura makumi matatu.

PCR tekinoroji yakapayona muna 1983 naKary Mullis weCetus, USA.Mullis akanyorera PCR patent muna 1985 uye akaburitsa bepa rekutanga rePCR rezvidzidzo paSainzi mugore rimwe chetero.Mullis akahwina Mubairo weNobel muChemistry muna 1993.

Basic Principles yePCR

PCR inogona kukudza zvimedu zveDNA zvakanangwa nekanopfuura miriyoni imwe.Nheyo yacho ndeyokuti pasi pecatalysis yeDNA polymerase, DNA yemubereki strand inoshandiswa se template, uye imwe primer inoshandiswa sekutanga kwekuwedzera.Inodzokororwa mu vitro kuburikidza nematanho akadai se denaturation, annealing, uye kuwedzera.Maitiro emwanasikana strand DNA inopindirana nemubereki strand template DNA.

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Iyo yakajairwa PCR maitiro akakamurwa kuita matanho matatu:

1. Denaturation: Shandisa tembiricha yakanyanya kuparadzanisa DNA mbiri tambo.Iyo hydrogen zvisungo pakati peDNA tambo mbiri dzakatyoka pakupisa kwakanyanya (93-98°C).

2. Annealing: Mushure mokunge DNA ine tambo mbiri yakaparadzaniswa, kutonhora kunoderedzwa kuitira kuti primer isungirire kune imwe-stranded DNA.

3. Kuwedzera: Iyo DNA polymerase inotanga kubatanidza tambo dzinopindirana pamwe chete netambo dzeDNA kubva kune yekutanga inosungwa kana tembiricha yadzikiswa.Kana kuwedzerwa kwapera, kutenderera kunopedzwa, uye nhamba yezvimedu zveDNA inopetwa kaviri.

Kudzorera matanho matatu aya 25-35 nguva, nhamba yezvimedu zveDNA ichawedzera zvakanyanya.

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Hungwaru hwePCR ndehwekuti maprimers akasiyana anogona kugadzirwa kune akasiyana anotangwa majini, kuitira kuti zvinonangwa zvimedu zvejeni zvinogona kukwidziridzwa munguva pfupi.

Parizvino, PCR inogona kukamurwa kuita zvikamu zvitatu, zvinoti zvakajairika PCR, fluorescent quantitative PCR uye digital PCR.

Chizvarwa chekutanga cheyakajairika PCR

Shandisa yakajairwa PCR amplification chiridzwa kukwidziridza jini rinotarirwa, wobva washandisa agarose gel electrophoresis kuona chigadzirwa, kuongororwa kwemhando chete kunogona kuitwa.

Izvo zvakanyanya kuipa kwechizvarwa chekutanga PCR:

-Inotarisana kune isiri-chaiyo amplification uye yenhema yakanaka mhedzisiro.

-Kuonekwa kunotora nguva yakareba uye kushanda kwakaoma.

-Chete yemhando yekuongorora inogona kuitwa.

Chechipiri-chizvarwa fluorescence quantitative PCR

Fluorescence quantitative PCR (Real-Time PCR), inozivikanwawo seqPCR, inoshandiswa kutarisa kuunganidzwa kwezvigadzirwa zvakasimudzwa kuburikidza nekuunganidzira masaini efluorescent nekuwedzera fluorescent probes iyo inogona kuratidza kufambira mberi kweiyo reaction system, uye kutonga mhedzisiro kuburikidza nefluorescence curve uye kukosha kwayo inogona kuve yakajairwa curve.

Nekuti tekinoroji yeqPCR inoitwa muhurongwa hwakavharwa, mukana wekusvibiswa wakaderedzwa, uye chiratidzo chefluorescence chinogona kutariswa kuti chionekwe kuwanda, saka ndiyo inonyanya kushandiswa mukurapa kwekiriniki uye yave iyo inotonga tekinoroji muPCR.

Izvo zvinhu zvefluorescent zvinoshandiswa mune chaiyo-nguva fluorescent quantitative PCR inogona kukamurwa kuita: TaqMan fluorescent probes, molecular beacon uye fluorescent dhayi.

1) TaqMan fluorescent probe:

Munguva yePCR kukwidziridzwa, imwe fluorescent probe inowedzerwa uchiwedzera peya yekutanga.Iyo probe ndeye oligonucleotide, uye iyo migumo miviri yakanyorwa zvakateerana nereporter fluorescent boka uye quencher fluorescent boka.

Kana iyo probe isina kusimba, chiratidzo chefluorescent chinoburitswa neboka renhau inotorwa neboka rekudzima;panguva yePCR amplification, iyo 5'-3 ′ exonuclease chiitiko cheTaq enzyme inotsemura uye kushatisa probe, ichiita mutori wenhau fluorescent boka uye quencher Boka refluorescent rakapatsanurwa, kuitira kuti iyo fluorescence monitoring system igamuchire iyo fluorescence chiratidzo, ndiko kuti, pese apo DNA strand inoumbwa, inosimudza fluorescent chiratidzo chakanyatso kuwiriraniswa nekuumbwa kwePCR chigadzirwa.

2) SYBR fluorescent dhayi:

MuPCR reaction system, yakawandisa yeSYBR fluorescent dhayi inowedzerwa.Mushure mekunge SYBR fluorescent dhayi isiri-yakanyanya kuisirwa muDNA kaviri-strand, inoburitsa chiratidzo chefluorescent.Iyo SYBR dhayi mamorekuru isina kuiswa muketani haizobudisi chero chiratidzo chefluorescent, nekudaro kuve nechokwadi chechiratidzo chefluorescent Kuwedzera kwePCR zvigadzirwa zvinonyatsoenderana nekuwedzera kwePCR zvigadzirwa.SYBR inongosungirira kuDNA yakasungwa kaviri, saka curve inonyunguduka inogona kushandiswa kuona kana kuita kwePCR kwakanangana.

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3) Molecular beacons

Iyo stem-loop yakapetwa kaviri-yakanyorwa oligonucleotide probe inoumba hairpin chimiro cheanosvika masere mabhesi ku5 uye 3 kumagumo.Iyo nucleic acid sequences kumagumo ese ari maviri anowirirana, zvichiita kuti boka refluorescent uye boka rekudzima rive rakaoma.Pedyo, haizobudisi fluorescence.

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Mushure mekunge chigadzirwa chePCR chagadzirwa, panguva yekuita annealing, chikamu chepakati cheiyo molecular beacon inopetwa neimwe DNA sequence, uye iyo fluorescent gene inoparadzaniswa kubva kuquencher gene kuti ibudise fluorescence.

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Izvo zvakanyanya kuipa kwechipiri-chizvarwa PCR:

Sensitivity ichiri kushaikwa, uye kutariswa kweyepasi-kopi sampuli hakuna kururama.

Pane kurudziro yekumashure, uye mhedzisiro yacho inogona kukanganisa.

Chizvarwa chechitatu digitaalinen PCR

Digital PCR (DigitalPCR, dPCR, Dig-PCR) inoverengera nhamba yekopi yekutevedzana kwechinangwa kuburikidza nekuona kwekupedzisira-nzvimbo, uye inogona kuita chaiyo yakakwana yehuwandu hwekuona pasina kushandisa mukati mekutonga uye yakajairwa curves.

Digital PCR inoshandisa endpoint yekuona uye haitsamiri pane Ct kukosha (kutenderera chikumbaridzo), saka iyo digital PCR reaction haina kukanganiswa neamplification kunyatsoshanda, uye kushivirira kune PCR reaction inhibitors inovandudzwa, nehupamhi hwechokwadi uye kuberekazve.

Nekuda kwehunhu hwekunzwa kwepamusoro uye kurongeka kwepamusoro, haipindire nyore nePCR reaction inhibitors, uye inokwanisa kuwana hwerengedzo yechokwadi yakakwana pasina zvigadzirwa zvakajairwa, izvo zvave kutsvaga uye kushandisa hotspot.

Zvinoenderana nemhando dzakasiyana dzeiyo reaction unit, inogona kukamurwa kuita matatu marudzi: microfluidic, chip uye droplet masisitimu.


Nguva yekutumira: Jul-08-2021