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Ọtụtụ mmepụta mgbanwe mgbanwe na akụkọ ihe mere eme nke teknụzụ nchọpụta n'uche m bụ teknụzụ immunolabeling dabere na ụkpụrụ nke njikọ antigen-antibody kpọmkwem, teknụzụ PCR na teknụzụ usoro.Taa, anyị ga-ekwu maka teknụzụ PCR.Dị ka evolushọn nke nkà na ụzụ PCR si dị, ndị mmadụ na-ekekarị nkà na ụzụ PCR n'ọgbọ atọ: nkà na ụzụ PCR nkịtị, ezigbo oge fluorescent quantitative PCR technology na digital PCR technology.

Cusoro PCR

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KARY MULLIS (1944.12.28-2019.8.7)

Kary Mullis chepụtara mmeghachi omume polymerase (polymerase chain reaction, PCR) na 1983. A na-ekwu na mgbe ọ na-akwọ enyi ya nwanyị, ọ na-enwe mmetụta ọkụ na mberede na echiche nke ụkpụrụ PCR (na uru nke ịkwọ ụgbọala).E nyere Kary Mullis ihe nrite Nobel na Chemistry na 1993. Akwụkwọ akụkọ New York Times kwuru, sị: “Ọ bụ nke mbụ na nke dị ịrịba ama, ọ fọrọ nke nta ka ọ na-ekesa bayoloji n'ime oge tupu PCR na post-PCR.

Ụkpụrụ nke PCR: N'okpuru catalysis nke DNA polymerase, nne strand DNA na-eji dị ka template, na kpọmkwem primer na-eji dị ka mmalite nke ndọtị, na nwa strand DNA nkwado nne strand template DNA na-depụtaghachiri na vitro site denaturation, annealing, ndọtị na ndị ọzọ nzọụkwụ.Ọ bụ teknụzụ mmụba nke DNA na vitro, nke nwere ike ịgbasa ngwa ngwa yana gbasie DNA ọ bụla lekwasịrị anya na vitro.

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Uru nke PCR nkịtị
1.Usoro kpochapụwo, ụkpụrụ zuru ụwa ọnụ na nke ụlọ
2.Obere ego nke reagent ngwá ọrụ
3.Enwere ike ị nwetaghachi ngwaahịa PCR maka nnwale ndị ọzọ dị ndụ
Igwe PCR Foregene akwadoro: https://www.foreivd.com/foreamp-sn-695-series-thermal-cycler-96-wells-pcr-machine-product/
Ngwaahịa ndị metụtara: https://www.foreivd.com/pcr-herotm-with-dye-product/
Ọdịmma nke PCR nkịtị
1.mfe imetọ
2.arụrụ arụrụ arụ
3.naanị qualitative analysis
4.Mmetụta na-agafeghị oke
5.Enwere nkwalite na-abụghị nke akọwapụtara, na mgbe band nke na-abụghị nke akọwapụtara bụ otu nha dị ka band nke ebumnuche, enweghị ike ịmata ya.
 
CPCR dabeere na apillary electrophoresis
Na nzaghachi na adịghị ike nke PCR nkịtị, ụfọdụ ndị na-emepụta ewepụtala ngwá ọrụ dabere na ụkpụrụ nke electrophoresis capillary.Nzọụkwụ electrophoresis ka emechara PCR n'ime capillary.Mmetụta dị elu dị elu, enwere ike ịmata ọdịiche dị n'ọtụtụ ntọala na mmụba nwere ike gbakọọ site na MAERKER.ọdịnaya ngwaahịa.Ọdịmma ya bụ na ngwaahịa PCR ka kwesịrị imeghe ma tinye ya n'ime ngwa, ma a ka nwere nnukwu ihe ize ndụ nke mmetọ.

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CapillaryElectrophoresis

 

2. teknụzụ fluorescent quantitative PCR (Quantitative Real-time PCR, qPCR) teknụzụ.Fluorescent quantitative PCR, nke a na-akpọkwa Real-Time PCR, bụ teknụzụ ọnụọgụ ọnụọgụ ọhụrụ nke PE (Perkin Elmer) mepụtara na 1995. Akụkọ mmepe nke fluorescent quantitative PCR bụ akụkọ ihe mere eme nke mgbagha mkpụrụ obi nke ndị dike dị ka ABI, Roche, na Bio-Rad.Ọ bụrụ na ị nwere mmasị, ị nwere ike lelee ya.Usoro a bụ usoro PCR kacha tozuru oke na nke a na-ejikarị eme ihe ugbu a.

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

Uzo esi esiji fluorescent (SYBR Green I):SYBR Green I bụ ihe a na-ejikarị DNA na-ejikọta maka PCR quantitative, nke jikọtara na-abụghị kpọmkwem na DNA nwere eriri abụọ.Na steeti nweere onwe ya, SYBR Green na-ewepụta fluorescence na-adịghị ike, mana ozugbo ejikọtara ya na DNA nwere eriri abụọ, fluorescence ya na-abawanye ugboro 1000.Ya mere, mkpokọta fluorescent mgbama na-ewepụta site na mmeghachi omume dabara na ọnụọgụ DNA nwere eriri abụọ dị ugbu a ga-abawanye site na mmụba nke ngwaahịa agbakwunyere.Ebe ọ bụ na agba agba na-ejikọtaghị kpọmkwem na DNA nwere eriri abụọ, enwere ike iwepụta nsonaazụ ụgha.

Ngwaahịa ndị metụtara: https://www.foreivd.com/real-time-pcr-easytm-sybr-green-i-kit-product/

Usoro nyocha fluorescent (teknụzụ Taqman): N'ogeNkwalite PCR, a na-agbakwunye nyocha fluorescent kpọmkwem n'otu oge ahụ dị ka otu ụzọ primers.Nyocha a bụ oligonucleotide linear, nwere otu ndị nta akụkọ fluorescent na otu fluorescent quencher otu nke akpọrọ na nsọtụ abụọ ahụ.Mgbe nyocha ahụ adịghị emebi emebi, mgbaàmà fluorescent nke ndị otu nta akụkọ na-ewepụta na-etinye uche na ya site na otu quencher, na nchọpụta ahụ Enweghị mgbaàmà fluorescent;n'oge PCR amplification (na mgbatị mgbatị), ọrụ 5'-3' Dicer nke Taq enzyme ga-agbaze ma mebie nyocha ahụ, nke mere na ndị na-ede akụkọ fluorescent group na ndị na-ekpo ọkụ na-ekpo ọkụ na-ekewapụ, nke mere na usoro nlekota fluorescence nwere ike ịnata mgbaàmà fluorescent, ya bụ, oge ọ bụla a yinye DNA na-amụbawanye, ihe mgbaàmà na-eme ka ọ bụrụ ihe mgbagwoju anya nke moleculation nke na-eme ka ọ bụrụ ihe mgbagwoju anya nke na-eme ka ọ bụrụ ihe mgbagwoju anya nke moleculation. s na nhazi nke ngwaahịa PCR.Usoro nyocha Taqman bụ usoro nchọpụta a na-ejikarị na nchọpụta ụlọ ọgwụ.

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

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Uru nke qPCR
1.Usoro a tozuru oke na akụrụngwa nkwado na reagents zuru ezu
2.Ọkara ọnụ nke reagents
3.mfe iji
4.Mmetụta nchọpụta dị elu na nkọwapụta
 
Ọdịmma nke qPCR

Mgbanwe nke mkpụrụ ndụ ihe mgbaru ọsọ na-eduga na nchọpụta efu.
Enweghị ike ikpebi nsonaazụ nke ndebiri ntinye uche dị ala.
Enwere nnukwu njehie mgbe ị na-eji usoro ọkọlọtọ maka nchọpụta ọnụọgụ.
 
3. Digital PCR (dijitalụ PCR, dPCR) teknụzụ
PCR dijitalụ bụ usoro maka nleba anya zuru oke nke molekul acid nucleic.E jiri ya tụnyere qPCR, PCR dijitalụ nwere ike gụọ ọnụọgụ DNA/RNA ozugbo, nke bụ ọnụọgụ nke mkpụrụ ndụ acid nucleic na mbido mbido.Na 1999, Bert Vogelstein na Kenneth W. Kin-zler kwadoro echiche nke dPCR.
 
Na 2006, Fluidigm bụ onye mbụ rụpụtara ngwa dPCR dabere na mgbawa azụmahịa.Na 2009, Life Teknụzụ bidoro sistemụ OpenArray na QuantStudio 12K Flex dPCR.Na 2013, Life Teknụzụ malitere usoro QuantStudio 3DdPCR, nke na-eji teknụzụ nanoscale microfluidic mgbawa dị elu iji kesaa ihe nlele na sel 20,000 n'otu n'otu.na mmeghachi omume nke ọma.

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N'afọ 2011, Bio-Rad weputara ngwa QX100 dPCR nke sitere na droplet, nke na-eji teknụzụ mmiri na mmanụ na-ekesa ihe nlele ahụ na mmanụ 20,000 nke mmiri mmiri n'ime mmanụ, ma na-eji nyocha mmiri nyocha iji nyochaa ụmụ irighiri mmiri.N'afọ 2012, RainDance weputara ngwa RainDrop dPCR, nke gas na-akpata nrụgide dị elu, kewaa usoro mmeghachi omume ọkọlọtọ ọ bụla n'ime emulsion mmeghachi omume nwere 1 nde na 10 nde picolite-larịị micro-droplets.

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Ka ọ dị ugbu a, PCR dijitalụ etolitela isi akụkụ abụọ, ụdị mgbawa na ụdị droplet.N'agbanyeghị ụdị PCR dijitalụ, ụkpụrụ ya bụ isi na-amachi dilution, njedebe PCR na nkesa Poisson.A na-ekewa usoro mmeghachi omume PCR ọkọlọtọ nke nwere ndebiri nucleic acid n'ime iri puku kwuru iri puku mmeghachi omume PCR, nke a na-ekesa na ibe ma ọ bụ microdroplets, nke mere na mmeghachi omume ọ bụla nwere ihe dị ka o kwere mee, molecule template, na a na-eme mmeghachi omume PCR otu-molecule.Site n'ịgụ fluorescence A na-agụta ọnụnọ ma ọ bụ enweghị mgbaàmà ahụ, a na-emekwa nha zuru oke mgbe emechara nkesa Poisson statistical.

Ndị a bụ njirimara nke ọtụtụ nyiwe PCR dijitalụ m jiworo:

1. Bio-Rad QX200 droplet dijitalụ PCR Bio-RadQX200 bụ ikpo okwu PCR dijitalụ nke ọma, usoro nchọpụta bụ isi: 20,000 n'omume na-emepụta site na droplet generator Water-in-oil micro-droplets na-abawanye na igwe PCR nkịtị, na n'ikpeazụ akara ngosi fluorescence nke micro-droplet ọ bụla na-agụ site na onye na-agụ micro-droplet.Ọrụ ahụ dị mgbagwoju anya karị, ihe ize ndụ nke mmetọ bụ ọkara.

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Xinyi TD1 micro-droplet dijitalụ PCRXinyi TD1 bụ ụlọ PCR dijitalụ dijitalụ, usoro nchọpụta bụ isi: mepụta 30,000-50,000 mmiri n'ime mmanụ site na generator droplet, gbasaa na ngwa PCR nkịtị, ma n'ikpeazụ gafere Onye na-agụ droplet na-agụ akara ngosi fluorescent nke ọ bụla droplet.A na-eme ma ọgbọ droplet na ịgụ ihe n'elu ikpo okwu a na mgbawa raara onwe ya nye nwere obere ihe ize ndụ nke mmetọ.

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 STILLA Naica micro-droplet mgbawa dijitalụ PCRSTILLA Naica bụ PCR dijitalụ ọhụrụ dị ọhụrụ.Usoro nchọpụta bụ isi bụ: tinye ihe ngwọta mmeghachi omume na mgbawa, tinye mgbawa n'ime usoro micro-droplet na nkwalite, wee mepụta 30,000 micro-droplets.Na-agbasa na mgbawa, na PCR amplification na-agwụ agwụ na mgbawa.Mgbe ahụ, a na-ebufe mgbawa a na-amụbawanye na usoro nyocha ọgụgụ micro-droplet, na-agụkwa mgbaàmà fluorescent site na ịse foto.Ebe ọ bụ na usoro ahụ dum na-ewere ọnọdụ na mgbawa mechiri emechi, ihe ize ndụ nke mmetọ dị ala.

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4. ThermoFisher QuantStudio 3D mgbawa dijitalụ PCR

ThermoFisher QuantStudio 3D bụ PCR dijitalụ dijitalụ ọzọ ama ama.Usoro nchọpụta ya bụ isi bụ: tinye ihe ngwọta mmeghachi omume n'ime onye na-agbasa, ma gbasaa ngwọta mmeghachi omume na mgbawa na 20,000 microwells site na mgbasa ozi., tinye mgbawa na igwe PCR ka ọ gbasaa, ma mesịa tinye mgbawa n'ime onye na-agụ ya wee see foto iji gụọ mgbaàmà fluorescent.Ọrụ ahụ dị mgbagwoju anya, a na-emekwa usoro ahụ dum na mgbawa mechiri emechi, ihe ize ndụ nke mmetọ dị ala.

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5. JN MEDSYS Clarity mgbawa dijitalụ PCR

JN MEDSYS Clarity bụ ụdị mgbawa ọhụrụ PCR dijitalụ.Usoro nchọpụta ya bụ isi bụ: tinye ngwọta mmeghachi omume n'ime ngwa ngwa, ma gbasaa ngwọta mmeghachi omume n'otu n'otu na tubes 10,000 PCR nke edobere na PCR tube site na ngwa ngwa.Na mgbawa microporous, mmeghachi omume mmeghachi omume na-abanye na mgbawa site na capillary action, na PCR tube na mgbawa na-etinye na PCR igwe maka amplification, na n'ikpeazụ a na-etinye mgbawa n'ime onye na-agụ ka ịgụ fluorescent mgbaàmà site na-ese foto.Ọrụ ahụ dị mgbagwoju anya karị.Ihe ize ndụ nke mmetọ dị ala.

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A na-achịkọta parampat nke ikpo okwu PCR dijitalụ ọ bụla dị ka ndị a:

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Ihe nleba anya nke igwe PCR dijitalụ bụ: ọnụ ọgụgụ nke nkeji nkewa, ọnụ ọgụgụ nke ọwa fluorescent, mgbagwoju anya nke ọrụ na ihe ize ndụ nke mmetọ.Ma ihe kacha mkpa bụ nchọpụta ziri ezi.Otu ụzọ isi nyochaa nyiwe PCR dijitalụ bụ iji ọtụtụ nyiwe PCR dijitalụ iji nyochaa ibe ha, na ụzọ ọzọ bụ iji ihe ọkọlọtọ nwere ụkpụrụ ziri ezi.

Uru nke dPCR
1.Inweta ọnụọgụgụ zuru oke
2.Mmetụta dị elu na nkọwapụta
3.Nwere ike ịchọpụta ụdị nnomi dị ala
Ọdịmma nke dPCR1. Ngwá ọrụ dị oke ọnụ na reagents 2. Mgbagwoju anya na-arụ ọrụ na ogologo oge nchọpụta 3. nso nso nchọpụta dị warara.

Ka ọ dị ugbu a, ọgbọ atọ nke teknụzụ PCR nwere uru na ọghọm nke ha, na nke ọ bụla nwere mpaghara ngwa nke ya, ọ bụghịkwa mmekọrịta nke otu ọgbọ na-anọchi nke ọzọ.Ọganihu teknụzụ na-aga n'ihu agbanyela ume ọhụrụ n'ime teknụzụ PCR, na-enyere ya aka imeghe otu ụzọ ngwa na-aga n'ihu, na-eme ka nchọpụta nucleic acid dịkwuo mfe na nke ziri ezi.
Isi mmalite: Dr. Yuan na-ewere gị maka ule
 
Ngwaahịa akwadoro:


Oge nzipu: Nov-18-2022