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Zviitiko zvakati wandei zveshanduko munhoroondo yekuona tekinoroji mupfungwa dzangu ndeye immunolabeling tekinoroji yakavakirwa pamusimboti weantigen-antibody chaiyo kusunga, PCR tekinoroji uye kuteedzera tekinoroji.Nhasi tichataura nezve PCR tekinoroji.Zvinoenderana nekushanduka kwePCR tekinoroji, vanhu vanogara vachipatsanura tekinoroji yePCR muzvizvarwa zvitatu: yakajairwa PCR tekinoroji, chaiyo-nguva fluorescent quantitative PCR tekinoroji uye digital PCR tekinoroji.

Common PCR nzira

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KARY MULLIS (1944.12.28-2019.8.7)

Kary Mullis akagadzira polymerase chain reaction (polymerase chain reaction, PCR) muna 1983. Zvinonzi paakanga achityaira musikana wake, akangoerekana ava nekupenya kwekufemerwa uye akafunga nezvemutemo wePCR (pamusoro pezvibatsiro zvekutyaira).Kary Mullis akapiwa Mubayiro weNobel muChemistry muna 1993. The New York Times yakatsinhira, kuti: “Yapakuvamba zvikuru uye inokosha, inodokuparadzanisa biology kuva pre-PCR uye post-PCR eras.

Nheyo yePCR: Pasi pecatalysis yeDNA polymerase, mai strand DNA inoshandiswa setemplate, uye iyo chaiyo primer inoshandiswa senzvimbo yekutanga yekuwedzera, uye mwanasikana strand DNA inopindirana naamai strand template DNA inokopwa mukati. vitro kuburikidza ne denaturation, annealing, kuwedzera uye mamwe matanho.Iyo DNA synthesis amplification tekinoroji in vitro, inogona kukurumidza uye nekunyatso kusimudzira chero chinangwa cheDNA in vitro.

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Zvakanakira zveyakajairwa PCR
1.Classic nzira, yakakwana yepasirese uye yemumba zviyero
2.Mutengo wakaderera wezviridzwa reagents
3.Zvigadzirwa zvePCR zvinogona kudzoserwa kune mamwe mamorekuru biology kuyedza
Inokurudzirwa Foregene PCR muchina: https://www.foreivd.com/foreamp-sn-695-series-thermal-cycler-96-wells-pcr-machine-product/
Zvigadzirwa zvinoenderana: https://www.foreivd.com/pcr-herotm-with-dye-product/
Kuipa kweyakajairika PCR
1.nyore kusvibisa
2.kushanda kwakaoma
3.chete qualitative analysis
4.Kunzwa kuri pakati nepakati
5.Iko kusiri-chaiyo amplification, uye kana iyo isiri-chaiyo bhendi yakaenzana neiyo bhendi inotarirwa, haigone kusiyaniswa.
 
Capillary electrophoresis-based PCR
Mukupindura kukanganisa kwePCR yakajairwa, vamwe vagadziri vakaunza zviridzwa zvinoenderana nemusimboti we capillary electrophoresis.Iyo electrophoresis nhanho mushure mePCR amplification yapedzwa mu capillary.Kunzwa kwakakwira, uye mutsauko wemabhesi akati wandei unogona kusiyaniswa uye iyo amplification inogona kuverengerwa neMAERKER.chigadzirwa chemukati.Izvo zvakashata ndezvekuti PCR chigadzirwa chichiri kuda kuvhurwa nekuiswa muchiridzwa, uye pachine njodzi huru yekusvibiswa.

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CapillaryElectrophoresis

 

2. Real-time fluorescent quantitative PCR (Quantitative Real-time PCR, qPCR) tekinorojiFluorescent quantitative PCR, inonziwo Real-Time PCR, ndiyo itsva nucleic acid quantitative tekinoroji yakagadziridzwa nePE (Perkin Elmer) muna 1995. Nhoroondo yebudiriro yefluorescent quantitative PCR inhoroondo yehondo dzinomutsa mweya dzehofori dzakaita seABI, Roche. , uye Bio-Rad.Kana uchifarira, unogona kuzvitarisa.Iyi tekinoroji parizvino ndiyo yakanyanya kukura uye inoshandiswa zvakanyanya semi-quantitative PCR maitiro.

Inokurudzirwa qPCR Machine:https://www.foreivd.com/mini-real-time-pcr-system-forequant-sf2sf4-product/

Fluorescent dhayi nzira (SYBR Green I):SYBR Green I ndiyo inonyanya kushandiswa DNA-inosunga dhayi yehuwandu PCR, inosunga isiri-chaiyo kuDNA ine tambo mbiri.Munzvimbo yakasununguka, SYBR Green inoburitsa isina kusimba fluorescence, asi kana yasungwa kaviri-yakasungwa DNA, fluorescence yayo inowedzera 1000-fold.Naizvozvo, iyo yakazara fluorescent siginecha inoburitswa nekuita inoenzana nehuwandu hwekaviri-yakasungwa DNA iripo uye ichawedzera nekuwedzera kwechigadzirwa chakawedzerwa.Sezvo dhayi inosunga isiri-chaiyo kune kaviri-yakasungwa DNA, yenhema mhedzisiro inogona kugadzirwa.

Zvigadzirwa zvinoenderana: https://www.foreivd.com/real-time-pcr-easytm-sybr-green-i-kit-product/

Fluorescent probe nzira (Taqman tekinoroji): PanguvaPCR amplification, yakananga fluorescent probe inowedzerwa panguva imwe chete sepairi yekutanga.Iyo probe ndeye mutsara oligonucleotide, ine fluorescent reporter boka uye fluorescent quencher boka zvakateerana zvakanyorwa pamigumo yese.Kana iyo probe isingasviki, chiratidzo chefluorescent chinobudiswa neboka remutori wenhau chinotorwa neboka re quencher, uye kuona Hapana chiratidzo chefluorescent;panguva yePCR amplification (muchikamu chekuwedzera), iyo 5'-3' Dicer basa reTaq enzyme inogaya uye kushatisa probe, kuitira kuti mutori wenhau fluorescent boka uye quencher fluorescent boka vaparadzane, kuitira kuti fluorescence monitoring system Iyo fluorescent. chiratidzo chinogona kugamuchirwa, ndiko kuti, pese painokwidziridzwa cheni yeDNA, molecule yefluorescent inoumbwa, iyo inoona kuwirirana kwakazara kwekuunganidzwa kwezviratidzo zvefluorescent uye kuumbwa kwePCR zvigadzirwa.Taqman probe nzira ndiyo inonyanya kushandiswa nzira yekuona mukuonekwa kwekiriniki.

Zvigadzirwa zvinoenderana: https://www.foreivd.com/quickeasy%e1%b5%80%e1%b4%b9-real-time-pcr-kit-taqman-product/

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Zvakanakira zve qPCR
1.Iyo nzira yakakura uye iyo inotsigira midziyo uye reagents yakakwana
2.Mari yepakati yemareagents
3.nyore kushandisa
4.High yekuona senitivity uye chaiyo
 
Zvakaipa zve qPCR

Kuchinja kwejini rinotarirwa kunotungamira pakupotsa kuonekwa.
Mhedzisiro yekuona ye-low-concentration template haigoni kutsanangurwa.
Pane mhosho hombe kana uchishandisa mwero curve yekuonekwa kwehuwandu.
 
3. Digital PCR (digital PCR, dPCR) teknolojia
Digital PCR inyanzvi yehuwandu hwehuwandu hwenyucleic acid mamorekuru.Kuenzaniswa neqPCR, digital PCR inogona kuverenga zvakananga huwandu hweDNA / RNA mamorekuru, inova ndiyo yakakwana quantification ye nucleic acid mamorekuru mune yekutanga sampu.Muna 1999, Bert Vogelstein naKenneth W. Kin-zler vakaronga zviri pamutemo pfungwa yedPCR.
 
Muna 2006, Fluidigm ndiyo yaive yekutanga kugadzira chip-based dPCR chiridzwa.Muna 2009, Life Technologies yakatanga OpenArray uye QuantStudio 12K Flex dPCR masisitimu.Muna 2013, Life Technologies yakatanga iyo QuantStudio 3DdPCR sisitimu, inoshandisa yakakwirira-density nanoscale microfluidic chip tekinoroji kugovera zvakaenzana masampuli kumaseru makumi maviri ega ega.mukuita zvakanaka.

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Muna 2011, Bio-Rad yakatanga donhwe-based QX100 dPCR chiridzwa, chinoshandisa mvura-mu-oiri tekinoroji kugovera zvakaenzana sample ku20,000 droplet-water-in-oiri, uye inoshandisa donhwe analyzer kuongorora madonhwe.Muna 2012, RainDance yakatanga iyo RainDrop dPCR chiridzwa, ichityairwa ne-high-pressure gasi, kuti igovane yega yega yega maitiro ekuita emulsion ine 1 miriyoni kusvika 10 miriyoni picoliter-level madiki madonhwe.

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Parizvino, digital PCR yakagadzira zvikwata zviviri zvikuru, chip type uye droplet type.Hazvina mhosva kuti rudzii rwePCR yedhijitari, misimboti yayo yakakosha inodzikamisa dilution, endpoint PCR uye Poisson kugovera.Iyo yakajairwa PCR reaction system ine nucleic acid templates yakakamurwa zvakaenzana kuita makumi ezviuru zvePCR maitiro, ayo anogoverwa kune machipisi kana mamicrodroplets, kuitira kuti imwe neimwe kuita kuine zvakawanda sezvinobvira template molecule, uye imwechete-morekuru template PCR reaction. kuitwa.Nokuverenga fluorescence Kuvapo kana kusavapo kwechiratidzo kunoverengwa, uye chiyero chakakwana chinoitwa mushure mekugadzirisa kwehuwandu hwekugoverwa kwePoisson.

Aya anotevera maitiro e akati wandei edhijitari PCR mapuratifomu andashandisa:

1. Bio-Rad QX200 droplet digital PCR Bio-RadQX200 ndeye yakasarudzika dhijitari PCR papuratifomu, yekutanga yekuona maitiro: zviuru makumi maviri zvemasamples anogadzirwa neinodonhedza jenareta Mvura-mu-oiri madiki-madonhwe anokwidziridzwa pamushini wakajairwa wePCR, uye pakupedzisira chiratidzo chefluorescence chegiro-donhwe rega rega rinoverengwa. ne micro-droplet reader.Kuvhiya kwacho kwakaoma, uye njodzi yekusvibiswa iri pakati.

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Xinyi TD1 micro-droplet digital PCRXinyi TD1 idhijitari PCR papuratifomu, iyo yekutanga yekuona maitiro: gadzira makumi matatu emvura-mu-mafuta madonhwe kuburikidza nemudonhwe jenareta, kusimudzira pane yakajairika PCR chiridzwa, uye pakupedzisira pfuura Muverengi wekudonha anoverenga chiratidzo chefluorescent chedonhwe rega rega.Zvese zviri zviviri chizvarwa chekudonhedza uye kuverenga mupuratifomu ino zvinoitwa mune yakatsaurirwa chip ine njodzi yakaderera yekusvibiswa.

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 STILLA Naica micro-droplet chip digital PCRSTILLA Naica ichangoburwa PCR chikuva.Iyo yekutanga yekuona maitiro ndeye: wedzera mhinduro mhinduro kune chip, isa chip muiyo micro-droplet chizvarwa uye amplification system, uye gadzira zviuru makumi matatu madiki madonhwe.Paridza pane chip, uye PCR kukwidziridzwa kunopedzwa pane chip.Ipapo iyo amplified chip inoendeswa kune iyo micro-droplet kuverenga yekuongorora system, uye chiratidzo chefluorescent chinoverengwa nekutora mifananidzo.Sezvo iyo yose nzira inoitika muvhavha chip, njodzi yekusvibiswa yakaderera.

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4. ThermoFisher QuantStudio 3D chip digital PCR

ThermoFisher QuantStudio 3D imwe yakasarudzika chip-yakavakirwa dijitari PCR chikuva.Yayo yekutanga yekuona maitiro ndeye: wedzera mhinduro mhinduro muparadzi, uye kuparadzira mhinduro mhinduro zvakaenzana pane chip ine 20,000 microwells kuburikidza nekuparadzira., isa chip pamushini wePCR kuti uwedzere, uye pakupedzisira isa chip muverengi uye tora mufananidzo kuti uverenge chiratidzo chefluorescent.Kushanda kwacho kwakaoma, uye nzira yose inoitwa muvhavha chip, uye njodzi yekusvibiswa yakaderera.

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5. JN MEDSYS Kujeka chip digital PCR

JN MEDSYS Kujeka inguva nyowani chip-mhando yedhijitari PCR chikuva.Yayo yekutanga yekuona maitiro ndeye: wedzera mhinduro mhinduro muapplicator, uye kuparadzira mhinduro mhinduro zvakaenzana pa10,000 PCR machubhu akaiswa muPCR chubhu kuburikidza nemushandisi.Paiyo microporous chip, mhinduro yekupindura inopinda muchip kuburikidza necapillary action, uye iyo PCR chubhu ine chip inoiswa pamushini wePCR wekusimudzira, uye pakupedzisira iyo chip inoiswa muverengi kuti averenge chiratidzo chefluorescent nekutora mufananidzo.Oparesheni yacho yakanyanya kuoma.Ngozi yekusvibiswa yakaderera.

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Iwo maparamita ega ega edhijitari PCR papuratifomu anopfupikiswa seizvi:

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Zviratidzo zvekuongorora zvedhijitari PCR papuratifomu ndeiyi: nhamba yezvikamu zvakapatsanurwa, huwandu hwemagetsi efluorescent, kuoma kwekushanda uye njodzi yekusvibiswa.Asi chinhu chinonyanya kukosha ndechekuona chokwadi.Imwe nzira yekuongorora digitaalinen PCR mapuratifomu ndeye kushandisa akawanda edhijitari PCR mapuratifomu kuonesa imwe neimwe, uye imwe nzira ndeyekushandisa zvakajairwa zvinhu zvine hunhu chaihwo.

Zvakanakira zve dPCR
1.Kuwana absolute quantification
2.Higher senitivity uye chaiyo
3.Inogona kuona yakaderera kopi samples
Zvakaipa zve dPCR1. Zvishandiso zvinodhura uye reagents 2. Kushanda kwakaoma uye nguva yakareba yekuona 3. Narrow yekuona mararamiro

Parizvino, zvizvarwa zvitatu zvePCR tekinoroji zvine zvazvakanakira uye zvakaipira, uye chimwe nechimwe chine minda yayo yekushandisa, uye haisi hukama kuti chimwe chizvarwa chinotsiva chimwe.Kuenderera mberi kwekufambira mberi kwetekinoroji kwakapinza hutsva hutsva muPCR tekinoroji, ichiigonesa kuvhura imwe nzira yekushandisa mushure meimwe, zvichiita kuti nucleic acid ionekwe zviri nyore uye zvakarurama.
Kwakabva: Dr. Yuan vanokuendesa kunoongororwa
 
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Nguva yekutumira: Nov-18-2022