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

PCR tekinoroji yakatangwa naKary Mullis wekuCetus, USA muna 1983. Mullis akanyorera PCR patent muna 1985 uye akaburitsa bepa rekutanga redzidzo rePCR reSainzi mugore rimwe chetero.Mullis akapihwa mubairo weNobel muchemistry muna 1993 nekuda kwebasa rake.

Basic Principles yePCR

PCR inogona kukudza zvimedu zveDNA zvakanangwa nekanopfuura miriyoni imwe.Iyo musimboti uri pasi pecatalysis yeDNA polymerase, uchishandisa mubereki strand DNA setemplate uye chaiyo primer senzvimbo yekutanga yekuwedzera.Inodzokororwa mu vitro kuburikidza nematanho akadai se denaturation, annealing, uye kuwedzera.Maitiro emwanasikana strand DNA inopindirana nemubereki strand template DNA.

Iyo yakajairwa PCR maitiro akakamurwa kuita matanho matatu:

1.Denaturation: Shandisa kupisa kwepamusoro kuti uparadzanise DNA kaviri tambo.Iyo hydrogen bond pakati peDNA tambo mbiri yakatyoka pakupisa kwakanyanya (93-98 ℃).

2.Annealing: Mushure mokunge DNA yakapetwa kaviri yakaparadzaniswa, kuderedza kutonhora kuitira kuti primer isungirire kune imwe-stranded DNA.

3.Kuwedzera: Iyo DNA polymerase inotanga kubatanidza tambo dzekuwedzera pamwe chete neDNA tambo 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.

Hungwaru hwePCR ndehwekuti maprimers akasiyana anogona kugadzirwa akasiyana anotangwa majini, kuitira kuti zvinonangwa zvimedu zvejeni zvigone 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:

1.Prone kune isiri-specific amplification uye nhema migumisiro yakanaka.

2.Kuona kunotora nguva yakareba uye kushanda kwakaoma.

3.Only qualitative test inogona kuitwa

Chechipiri-chizvarwa Real-Nguva PCR

Real-Time PCR, inozivikanwawo seqPCR, inoshandisa fluorescent probes inogona kuratidza kufambira mberi kweiyo reaction system, uye inotarisisa kuunganidzwa kwezvigadzirwa zvakakwidziridzwa kuburikidza nekuunganidzwa kwemasaini efluorescent, uye inotonga mhedzisiro kuburikidza nefluorescence curve.Inogona kuverengerwa nerubatsiro rweCq kukosha uye 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 probe, molecular beacon uye fluorescent dhayi.

1) TaqMan fluorescent probe:

Munguva yePCR kukwidziridzwa, imwe fluorescent probe inowedzerwa uchiwedzera peya yekutanga.Iyo probe ndeye oligonucleotide, uye migumo miviri yakanyorwa 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.

3) Molecular beacon:

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, hapana fluorescence ichagadzirwa.

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.

Izvo zvakanyanya kuipa kwechipiri-chizvarwa PCR:

Sensitivity ichiri kushaikwa, uye kuonekwa kweyepasi-kopi mienzaniso haina kururama.

Iko kune simba rekumashure kukosha, uye mhedzisiro inogona kukanganisa kukanganisa.

Kana paine PCR inhibitors mune reaction system, mhedzisiro yekuona 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 magumo ekuona uye haina kutsamira pane Ct kukosha (kutenderera chikumbaridzo), saka iyo digital PCR reaction haina kukanganiswa neamplification kunyatsoshanda, uye kushivirira kune PCR reaction inhibitors inovandudzwa, nehupamhi hwepamusoro 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 makuru marudzi: microfluidic, chip uye droplet masisitimu.

1) Microfluidic digital PCR, mdPCR:

Zvichienderana neiyo microfluidic tekinoroji, iyo DNA template inoparadzaniswa.Iyo microfluidic tekinoroji inogona kuona iyo sampuli nano-kusimudzira kana chizvarwa chemadonhwe madiki, asi madonhwe anoda yakakosha adsorption nzira uye obva asanganiswa nePCR reaction system.mdPCR zvishoma nezvishoma yakagamuchirwa nedzimwe nzira kutsiva.

2) Droplet-based digital PCR, ddPCR:

Shandisa tekinoroji yekugadzira mvura-mu-oiri kugadzira sampu kuita madonhwe, uye patsanura maitiro ekuita ane nucleic acid mamorekuru kuita zviuru zvenanoscale madonhwe, imwe neimwe isina nucleic acid inonangwa molecule kuti ionekwe, kana Iine imwe kune akati wandei nucleic acid inonangwa mamorekuru anoedzwa.

3) Chip-based digital PCR, cdPCR:

Shandisa iyo yakasanganiswa fluid nzira tekinoroji kunyora akawanda mamicrotubes uye microcavities pasilicon wafers kana quartz girazi, uye kudzora kuyerera kwemhinduro kuburikidza neakasiyana ekudzora mavharuvhu, uye patsanura mvura yemuenzaniso mumananometers ehukuru hwakafanana mumatsime ekuita edijitari PCR Reaction kuti uwane yakakwana quantification.

Izvo zvakanyanya kuipa kwechitatu chizvarwa PCR:

Zvishandiso uye reagents zvinodhura.

Izvo zvinodiwa zvemhando yetemplate zvakakwirira.Kana iyo template yakawanda ichipfuura iyo microsystem quantity, hazvizogoneke kuverengera, uye kana iri diki, iyo quantification yakaringana ichaderedzwa.

Nhema dzenhema dzinogona zvakare kugadzirwa kana paine isiri-chaiyo amplification.