Zosintha zingapo m'mbiri yaukadaulo wozindikira m'malingaliro mwanga ndiukadaulo wa immunolabeling potengera mfundo yomangiriza ma antigen-antibody, ukadaulo wa PCR ndi ukadaulo wotsatizana.Lero tikambirana zaukadaulo wa PCR.Malinga ndi kusinthika kwaukadaulo wa PCR, anthu amakonda kugawa ukadaulo wa PCR m'mibadwo itatu: ukadaulo wamba wa PCR, ukadaulo wanthawi yeniyeni wa fulorosenti wa PCR ndiukadaulo wa digito wa PCR.
Cnjira ya ommon PCR
KARY MULLIS (1944.12.28-2019.8.7)
Kary Mullis anapanga polymerase chain reaction (polymerase chain reaction , PCR) mu 1983. Zimanenedwa kuti pamene ankayendetsa bwenzi lake, mwadzidzidzi adakhala ndi kudzoza komanso kuganizira mfundo ya PCR (pa ubwino woyendetsa galimoto).Kary Mullis anapatsidwa Mphotho ya Nobel mu Chemistry mu 1993. The New York Times inathirira ndemanga kuti: “Yachikale kwambiri ndi yofunika kwambiri, yotsala pang’ono kugaŵa biology m’nyengo ya PCR ndi pambuyo pa PCR.
Mfundo ya PCR: Pansi pa catalysis ya DNA polymerase, DNA ya strand ya amayi imagwiritsidwa ntchito ngati template, ndipo choyambira chenichenicho chimagwiritsidwa ntchito ngati poyambira kutambasula, ndipo mwana wamkazi wa strand DNA wothandizana ndi DNA ya strand template ya DNA imakopera mu m'galasi kudzera mu denaturation, annealing, kutambasuka ndi masitepe ena.Ndi ukadaulo wa DNA synthesis amplification in vitro, womwe ungathe kukulitsa mwachangu komanso mwachindunji chandamale chilichonse cha DNA mu vitro.
Ubwino wa PCR wamba
1.Njira yachikale, miyezo yathunthu yapadziko lonse lapansi komanso yapakhomo
2.Mtengo wotsika wa reagents zida
3.Zogulitsa za PCR zitha kupezedwanso pazoyeserera zina zama cell biology
Makina ovomerezeka a Foregene PCR: https://www.foreivd.com/foreamp-sn-695-series-thermal-cycler-96-wells-pcr-machine-product/
Zogwirizana nazo: https://www.foreivd.com/pcr-herotm-with-dye-product/
Zoyipa za PCR wamba
1.zosavuta kuipitsa
2.ntchito yovuta
3.kokha Mkhalidwe kusanthula
4.Kukhudzika kwapakati
5.Pali kukulitsa kosakhazikika, ndipo gulu losakhala lachindunji likakhala lofanana ndi gulu lomwe mukufuna, silingasiyanitsidwe.
Capillary electrophoresis-based PCR
Poyankha zolakwa za PCR wamba, opanga ena adayambitsa zida zochokera ku mfundo ya capillary electrophoresis.Gawo la electrophoresis pambuyo pakukulitsa kwa PCR kumalizidwa mu capillary.Kukhudzika ndikwambiri, ndipo kusiyana kwa maziko angapo kumatha kusiyanitsa ndipo kukulitsa kumatha kuwerengedwa ndi MAERKER.mankhwala okhutira.Choyipa ndichakuti chipangizo cha PCR chikufunikabe kutsegulidwa ndikuyika mu chidacho, ndipo pakadali chiwopsezo chachikulu choipitsidwa.
CapillaryElectrophoresis
2. Ukadaulo weniweni wa fulorosenti wochulukirachulukira wa PCR (Quantitative Real-time PCR, qPCR)Fluorescent quantitative PCR, yomwe imatchedwanso Real-Time PCR, ndiukadaulo watsopano wa nucleic acid quantitative wopangidwa ndi PE (Perkin Elmer) mu 1995. Mbiri yachitukuko cha fluorescent quantitative PCR ndi mbiri ya kulimbana kolimbikitsa moyo kwa zimphona monga ABI, Roche, ndi Bio-Rad.Ngati mukufuna, mutha kuyang'ana.Njirayi pakadali pano ndiyo njira yokhwima kwambiri komanso yogwiritsidwa ntchito kwambiri pa PCR.
Makina ovomerezeka a qPCR: https://www.foreivd.com/mini-real-time-pcr-system-forequant-sf2sf4-product/
Njira ya utoto wa fluorescent (SYBR Green I):SYBR Green I ndi utoto womwe umagwiritsidwa ntchito kwambiri ndi DNA wa PCR wochulukira, womwe umamanga mosagwirizana ndi DNA yazingwe ziwiri.M'malo aulere, SYBR Green imatulutsa fluorescence yofooka, koma ikamangidwa ku DNA yazingwe ziwiri, fluorescence yake imachulukitsa ka 1000.Chifukwa chake, siginecha yonse ya fulorosenti yomwe imatulutsidwa ndi zomwe zimachitika imayenderana ndi kuchuluka kwa DNA yamitundu iwiri yomwe ilipo ndipo imachulukirachulukira pakuwonjezeka kwazinthu zokulitsa.Popeza utoto umamangiriza osati mwachindunji ku DNA yamitundu iwiri, zotsatira zabodza zitha kupangidwa.
Zogwirizana nazo: https://www.foreivd.com/real-time-pcr-easytm-sybr-green-i-kit-product/
Njira yofufuzira fulorosenti (ukadaulo wa Taqman): NthawiKukulitsa kwa PCR, kafukufuku wina wa fulorosenti amawonjezeredwa nthawi yomweyo ngati zoyambira.Kafukufukuyu ndi oligonucleotide yozungulira, yomwe ili ndi gulu la mtolankhani wa fulorosenti ndi gulu la fluorescent quencher motsatana zolembedwa mbali zonse ziwiri.Pamene kafukufukuyo ali bwino, chizindikiro cha fulorosenti choperekedwa ndi gulu la mtolankhani chimatengedwa ndi gulu la quencher, ndi kuzindikira Palibe chizindikiro cha fulorosenti;pa PCR amplification (mu siteji yowonjezera), ntchito ya 5'-3' Dicer ya Taq enzyme idzagaya ndi kusokoneza kafukufuku, kuti mtolankhani fulorosenti gulu ndi quencher fulorosenti gulu analekanitsidwa, kuti fulorosenti polojekiti dongosolo Chizindikiro fulorosenti akhoza kulandiridwa, ndiko kuti, nthawi iliyonse DNA molekyulu kuzindikira kuti unyolo wa DNA wapangidwa ndi fluorescent unyolo ndi fluorescent. kuchuluka kwa ma siginecha a fulorosenti ndikupanga zinthu za PCR.Njira ya Taqman probe ndiyo njira yomwe imagwiritsidwa ntchito kwambiri pozindikira zachipatala.
Zogwirizana nazo: https://www.foreivd.com/quickeasy%e1%b5%80%e1%b4%b9-real-time-pcr-kit-taqman-product/
Ubwino wa qPCR
1.Njirayi ndi yokhwima ndipo zida zothandizira ndi ma reagents zatha
2.Mtengo wapakati wa reagents
3.yosavuta kugwiritsa ntchito
4.Kuzindikira kwakukulu komanso kutsimikizika
Zoyipa za qPCR
Kusintha kwa jini yomwe mukufuna kumabweretsa kuphonya.
Chotsatira chodziwika cha template yotsika kwambiri sichingadziwike.
Pali cholakwika chachikulu mukamagwiritsa ntchito njira yokhotakhota kuti muzindikire kuchuluka kwake.
3. Digital PCR (digital PCR, dPCR) luso
Digital PCR ndi njira yowerengera mtheradi wa ma nucleic acid mamolekyulu.Poyerekeza ndi qPCR, digito PCR imatha kuwerenga mwachindunji kuchuluka kwa mamolekyu a DNA/RNA, omwe ndi kuchuluka kwathunthu kwa mamolekyu a nucleic acid pachitsanzo choyambirira.Mu 1999, Bert Vogelstein ndi Kenneth W. Kin-zler adapereka lingaliro la dPCR.
Mu 2006, Fluidigm inali yoyamba kupanga chida cha dPCR chopangidwa ndi chip.Mu 2009, Life Technologies idakhazikitsa OpenArray ndi QuantStudio 12K Flex dPCR machitidwe.Mu 2013, Life Technologies inayambitsa dongosolo la QuantStudio 3DdPCR, lomwe limagwiritsa ntchito luso lapamwamba la nanoscale microfluidic chip kugawa mofanana zitsanzo ku maselo 20,000.mukuchita bwino.
Mu 2011, Bio-Rad idakhazikitsa chida chochokera ku dontho la QX100 dPCR, chomwe chimagwiritsa ntchito ukadaulo wamadzi mumafuta kugawa mofananamo zitsanzo ku 20,000 droplet-water-in-mafuta, ndikugwiritsa ntchito makina osanthula madontho kusanthula madonthowo.Mu 2012, RainDance inayambitsa chida cha RainDrop dPCR, choyendetsedwa ndi mpweya wothamanga kwambiri, kugawaniza dongosolo lililonse la machitidwe mu emulsion yomwe ili ndi 1 miliyoni mpaka 10 miliyoni picoliter-level micro-droplets.
Pakadali pano, PCR ya digito yapanga magulu awiri akulu, mtundu wa chip ndi mtundu wa droplet.Ziribe kanthu kuti PCR ya digito yamtundu wanji, mfundo zake zazikulu ndikuchepetsa kuchepetsedwa, PCR yomaliza ndi kugawa kwa Poisson.Mawonekedwe a PCR omwe ali ndi ma templates a nucleic acid amagawidwa mofanana mu makumi zikwi za machitidwe a PCR, omwe amagawidwa ku tchipisi kapena ma microdroplets, kotero kuti zomwe zimachitika zimakhala ndi momwe zingathere ndi molekyulu ya template, ndipo template ya PCR imachitika.Powerenga fluorescence Kukhalapo kapena kusowa kwa chizindikiro kumawerengedwa, ndipo kuwerengera kwathunthu kumachitika pambuyo pa kuwerengetsa kwa chiwerengero cha Poisson.
Zotsatirazi ndi mawonekedwe a nsanja zingapo za digito za PCR zomwe ndagwiritsa ntchito:
1. Bio-Rad QX200 droplet digito PCR Bio-RadQX200 ndi nsanja yapamwamba kwambiri ya digito ya PCR, njira yodziwikiratu: Zitsanzo 20,000 zimapangidwa ndi jenereta ya droplet Madontho ang'onoang'ono amadzi mumafuta amakulitsidwa pamakina wamba a PCR, ndipo pamapeto pake chizindikiro cha fluorescence cha dontho lililonse laling'ono limawerengedwa ndi wowerenga madontho ochepa.Ntchitoyi ndi yovuta kwambiri, ndipo chiopsezo cha kuipitsa ndi chapakati.
Xinyi TD1 yaying'ono-droplet digito PCRXinyi TD1 ndi nsanja yapakhomo ya PCR yapanyumba, njira yodziwikiratu: imapanga madontho 30,000-50,000 amadzi-mu-mafuta kudzera mu jenereta ya madontho, kukulitsa chida chodziwika bwino cha PCR, ndipo pamapeto pake amadutsa Wowerenga madontho amawerenga chizindikiro cha fulorosenti cha dontho lililonse.Kupanga madontho komanso kuwerenga papulatifomu iyi kumachitika mu chipangizo chodzipatulira chokhala ndi chiwopsezo chochepa cha kuipitsidwa.
STILLA Naica micro-droplet chip digito PCRSTILLA Naica ndi nsanja yatsopano ya digito ya PCR.Njira yodziwikiratu ndi: onjezani yankho la chip, ikani chip mum'badwo wa madontho ang'onoang'ono ndi makina okulitsa, ndikupanga madontho 30,000 ang'onoang'ono.Kufalikira pa chip, ndipo kukulitsa kwa PCR kumatsirizidwa pa chip.Kenako chip chokulitsa chimasamutsidwa ku kachipangizo kakang'ono kakuwerengera kadontho kakang'ono, ndipo chizindikiro cha fulorosenti chimawerengedwa pojambula zithunzi.Popeza kuti ndondomeko yonseyi imachitika mu chip chotsekedwa, chiopsezo cha kuipitsidwa ndi chochepa.
4. ThermoFisher QuantStudio 3D chip digito PCR
ThermoFisher QuantStudio 3D ndi nsanja ina ya PCR yachikale ya digito.Njira yake yodziwikiratu ndi: yonjezerani yankho la momwe mungayankhire mu chofalitsa, ndikufalitsa njira yothetsera vutoli mofanana pa chip ndi ma microwell 20,000 kupyolera mu chofalitsa., ikani chip pamakina a PCR kuti mukulitse, ndipo potsiriza ikani chip mu owerenga ndikujambula chithunzi kuti muwerenge chizindikiro cha fulorosenti.Opaleshoniyo ndi yovuta kwambiri, ndipo ndondomeko yonseyi ikuchitika mu chip chotsekedwa, ndipo chiopsezo cha kuipitsidwa ndi chochepa.
5. JN MEDSYS Clarity chip digito PCR
JN MEDSYS Clarity ndi nsanja yatsopano yamtundu wa digito ya PCR.Njira yake yodziwikiratu ndi: yonjezerani yankho la zomwe mukugwiritsa ntchito, ndikufalitsa yankho lofananalo pamachubu 10,000 a PCR okhazikika mu chubu cha PCR kudzera pa chopaka.Pa chipangizo cha microporous, njira yothetsera vutoli imalowa mu chip kudzera mu capillary action, ndipo chubu cha PCR chokhala ndi chip chimayikidwa pa makina a PCR kuti akulitse, ndipo pamapeto pake chipcho chimayikidwa mu owerenga kuti awerenge chizindikiro cha fulorosenti pojambula chithunzi.Ntchitoyi ndi yovuta kwambiri.Chiwopsezo cha kuipitsidwa ndi chochepa.
Magawo a nsanja iliyonse ya digito ya PCR amafupikitsidwa motere:
Zizindikiro zowunikira papulatifomu ya digito ya PCR ndi: kuchuluka kwa magawo ogawanika, kuchuluka kwa mayendedwe a fulorosenti, zovuta zogwirira ntchito komanso kuopsa kwa kuipitsidwa.Koma chofunika kwambiri ndi kuzindikira kolondola.Njira imodzi yowunikira mapulaneti a digito a PCR ndikugwiritsa ntchito nsanja zingapo za digito za PCR kutsimikizirana, ndipo njira ina ndikugwiritsa ntchito zinthu zomwe zili ndi mfundo zolondola.
Ubwino wa dPCR
1.Kukwaniritsa quantification mtheradi
2.Kukhudzika kwakukulu ndi kutsimikizika
3.Mutha kuzindikira zitsanzo zochepa
Zoyipa za dPCR1. Zida zokwera mtengo ndi zopangira 2. Ntchito zovuta komanso nthawi yayitali yodziwikiratu 3. Kuzindikira kocheperako
Pakalipano, mibadwo itatu ya teknoloji ya PCR ili ndi ubwino ndi zovuta zawo, ndipo aliyense ali ndi minda yake yogwiritsira ntchito, ndipo si ubale umene mbadwo umodzi umalowa m'malo mwa wina.Kupita patsogolo kwaukadaulo kwadzetsa nyonga yatsopano muukadaulo wa PCR, kupangitsa kuti itsegule njira imodzi yolumikizirana, ndikupangitsa kuzindikira kwa nucleic acid kukhala kosavuta komanso kolondola.
Gwero: Dr. Yuan amakutengerani kukayezetsa
Zofunika:
Nthawi yotumiza: Nov-18-2022
