ʻO kekahi mau mea hou i loko o ka mōʻaukala o ka ʻenehana ʻike i loko o koʻu noʻonoʻo he ʻenehana immunolabeling e pili ana i ke kumu o ka paʻa pono antigen-antibody, ʻenehana PCR a me ka ʻenehana sequencing.I kēia lā e kamaʻilio mākou e pili ana i ka ʻenehana PCR.E like me ka ulu ʻana o ka ʻenehana PCR, hoʻokaʻawale nā kānaka i ka ʻenehana PCR i ʻekolu mau hanauna: ʻenehana PCR maʻamau, ʻenehana PCR fluorescent quantitative a me ʻenehana PCR kikohoʻe.
Cʻenehana PCR ommon
KARY MULLIS (1944.12.28-2019.8.7)
Ua hoʻokumu ʻo Kary Mullis i ka hopena o ke kaulahao polymerase (polymerase chain reaction, PCR) i ka makahiki 1983. Ua ʻōlelo ʻia i ka wā e hoʻokele ana ʻo ia i kāna hoaaloha, ua loaʻa koke iā ia kahi uila o ka hoʻoulu ʻana a noʻonoʻo i ke kumu o ka PCR (ma nā pono o ka hoʻokele).Ua hāʻawi ʻia ʻo Kary Mullis i ka Nobel Prize in Chemistry i ka makahiki 1993. Ua ʻōlelo ka New York Times: "He kumu nui a koʻikoʻi, kokoke e hoʻokaʻawale i ka biology i nā wā pre-PCR a me ka post-PCR.
ʻO ke kumu o ka PCR: Ma lalo o ka catalysis o ka DNA polymerase, ua hoʻohana ʻia ka DNA strand makuahine ma ke ʻano he kumu hoʻohālike, a ua hoʻohana ʻia ka primer kikoʻī ma ke ʻano he hoʻomaka o ka hoʻonui ʻana, a ua kope ʻia ke kaula DNA i hoʻohui ʻia i ka DNA template strand makuahine ma o ka denaturation, annealing, extension a me nā hana ʻē aʻe.He DNA synthesis amplification technology in vitro, hiki iā ia ke hoʻonui a hoʻonui i kekahi DNA target in vitro.
Nā pōmaikaʻi o ka PCR maʻamau
1.ʻO ke ala maʻamau, piha nā kūlana honua a me ka home
2.ʻO ke kumu kūʻai haʻahaʻa o nā mea hana reagents
3.Hiki ke kiʻi hou ʻia nā huahana PCR no nā hoʻokolohua molecular biology
Manaʻo ʻia ka mīkini PCR Foregene: https://www.foreivd.com/foreamp-sn-695-series-thermal-cycler-96-wells-pcr-machine-product/
Nā huahana pili: https://www.foreivd.com/pcr-herotm-with-dye-product/
Nā hemahema o ka PCR maʻamau
1.oluolu e hoohaumia
2.hana paʻakikī
3.ka nānā ʻana qualitative wale nō
4.ʻAno haʻahaʻa
5.Aia ka hoʻonui ʻole kikoʻī, a inā like ka nui o ka pūʻulu kikoʻī ʻole me ka pūʻali i hoʻopaʻa ʻia, ʻaʻole hiki ke ʻike ʻia.
CApillary electrophoresis-based PCR
I ka pane ʻana i nā hemahema o ka PCR maʻamau, ua hoʻolauna kekahi mau mea hana i nā mea kani e pili ana i ke kumu o ka electrophoresis capillary.ʻO ka hana electrophoresis ma hope o ka hoʻonui ʻana o PCR i ka capillary.ʻOi aku ka kiʻekiʻe o ka naʻau, a hiki ke ʻike ʻia ka ʻokoʻa o kekahi mau kumu a hiki ke helu ʻia ka amplification e MAERKER.maʻiʻo huahana.ʻO ka hemahema ʻo ia ka pono e wehe ʻia ka huahana PCR a hoʻokomo ʻia i loko o ka mea hana, a aia kekahi pilikia nui o ka hoʻohaumia ʻana.
CapillaryElectrophoresis
2. ʻenehana PCR (Quantitative Real-time PCR, qPCR)ʻO Fluorescent quantitative PCR, i kapa ʻia ʻo Real-Time PCR, he ʻenehana hou nucleic acid quantitative i hoʻomohala ʻia e PE (Perkin Elmer) i ka makahiki 1995. ʻO ka mōʻaukala hoʻomohala o ka fluorescent quantitative PCR he moʻolelo o nā hakakā ʻuhane o nā pilikua e like me ABI, Roche, a me Bio-Rad.Inā makemake ʻoe, hiki iā ʻoe ke nānā iā ia.ʻO kēia ʻenehana i kēia manawa ka ʻenehana semi-quantitative PCR ʻoi loa a hoʻohana nui ʻia.
Manaʻo ʻia ka mīkini qPCR: https://www.foreivd.com/mini-real-time-pcr-system-forequant-sf2sf4-product/
ʻO ke ʻano o ka waihoʻoluʻu fluorescent (SYBR Green I):ʻO SYBR Green I ka mea hoʻohana maʻamau i hoʻohana ʻia i ka DNA-binding dye no ka PCR quantitative, kahi e hoʻopaʻa ʻole ai i ka DNA stranded pālua.Ma ka moku'āina kūʻokoʻa, hoʻopuka ka SYBR Green i ka fluorescence nāwaliwali, akā i ka manawa i hoʻopaʻa ʻia i ka DNA stranded pālua, piʻi ka fluorescence i 1000-paila.No laila, ʻo ka hōʻailona fluorescent holoʻokoʻa i hoʻokuʻu ʻia e kahi pane e like me ka nui o ka DNA stranded pālua i kēia manawa a e hoʻonui ʻia me ka hoʻonui ʻana o ka huahana amplified.No ka hoʻopaʻa ʻole ʻana o ke kala i ka DNA kaulalua, hiki ke hana ʻia nā hopena maikaʻi ʻole.
Nā huahana pili: https://www.foreivd.com/real-time-pcr-easytm-sybr-green-i-kit-product/
ʻO ke kaʻina hana ʻimi Fluorescent (ʻenehana Taqman): I ka wāHoʻonui ʻia ka PCR, ua hoʻohui ʻia kahi ʻimi fluorescent kikoʻī i ka manawa like me nā kumu mua.He oligonucleotide linear ka probe, me ka pūʻulu mea hōʻike fluorescent a me ka pūʻulu quencher fluorescent i hōʻailona ʻia ma nā ʻaoʻao ʻelua.I ka wā e paʻa ai ka ʻimi, ʻo ka hōʻailona fluorescent i hoʻopuka ʻia e ka hui mea hoʻolaha e hoʻopili ʻia e ka hui quencher, a me ka ʻike ʻAʻohe hōʻailona fluorescent;i ka wā o ka hoʻonui ʻana o PCR (ma ka pae hoʻonui), ʻo ka hana 5'-3' Dicer o Taq enzyme e ʻeli a hoʻohaʻahaʻa i ka ʻimi, no laila e hoʻokaʻawale ʻia ka pūʻulu fluorescent a me ka hui fluorescent quencher, i hiki ke loaʻa ka hōʻailona fluorescent. ka hoʻokumu ʻana o nā huahana PCR.ʻO ke ʻano hana ʻimi ʻo Taqman ke ʻano ʻike maʻamau i hoʻohana ʻia i ka ʻike lapaʻau.
Nā huahana pili: https://www.foreivd.com/quickeasy%e1%b5%80%e1%b4%b9-real-time-pcr-kit-taqman-product/
Nā pōmaikaʻi o qPCR
1.Ua oo ke ala a ua piha na mea kokua a me na reagents
2.ʻO ke kumu kūʻai liʻiliʻi o nā reagents
3.oluolu e hoohana
4.ʻO ka ʻike kiʻekiʻe a me ka kikoʻī
Nā hemahema o ka qPCR
ʻO ka hoʻololi ʻana o ka gene i manaʻo ʻia e alakaʻi i ka ʻike nalo.
ʻAʻole hiki ke hoʻoholo ʻia ka hopena ʻike o ka laʻana haʻahaʻa.
Aia ka hewa nui i ka wā e hoʻohana ai i ka pihi maʻamau no ka ʻike quantitative.
3. Kikohoʻe PCR (kikohoʻe PCR, dPCR).
ʻO ka PCR kikohoʻe he ʻenehana no ka helu pono ʻana o nā molekole nucleic acid.Ke hoʻohālikelike ʻia me qPCR, hiki i ka PCR kikohoʻe ke heluhelu pololei i ka helu o nā molekala DNA/RNA, ʻo ia ka helu piha o nā molekala nucleic acid i ka laʻana hoʻomaka.I ka makahiki 1999, ua hoʻolauna mua ʻo Bert Vogelstein lāua ʻo Kenneth W. Kin-zler i ka manaʻo o ka dPCR.
I ka makahiki 2006, ʻo Fluidigm ka mea mua i hana i kahi mea hana dPCR e pili ana i ka chip.I ka makahiki 2009, ua hoʻokumu ʻo Life Technologies i nā ʻōnaehana OpenArray a me QuantStudio 12K Flex dPCR.I ka makahiki 2013, ua hoʻokumu ʻo Life Technologies i ka ʻōnaehana QuantStudio 3DdPCR, e hoʻohana ana i ka ʻenehana chip microfluidic chip nanoscale kiʻekiʻe e puʻunaue like i nā laʻana i 20,000 mau keena pākahi.i loko o ka hopena maikaʻi.
I ka makahiki 2011, ua hoʻokumu ʻo Bio-Rad i ka mea hana QX100 dPCR e hoʻohana ana i ka ʻenehana wai-i-ʻaila e puʻunaue like i ka hāpana i 20,000 droplet-water-in-oil, a hoʻohana i ka droplet analyzer e nānā i nā droplets.I ka makahiki 2012, ua hoʻokumu ʻo RainDance i ka mea kani RainDrop dPCR, i alakaʻi ʻia e ke kinoea piʻi kiʻekiʻe, e puʻunaue i kēlā me kēia ʻōnaehana pane maʻamau i loko o kahi emulsion hopena i loaʻa ka 1 miliona a 10 miliona picoliter-level micro-droplets.
I kēia manawa, ua hoʻokumu ʻo PCR digital i ʻelua mau ʻāpana nui, ʻano chip a me ke ʻano droplet.ʻAʻohe ʻano o ka PCR kikohoʻe, ʻo kāna mau kumu kumu e kaupalena ana i ka dilution, endpoint PCR a me Poisson distribution.ʻO ka ʻōnaehana PCR maʻamau i loaʻa nā ʻāpana nucleic acid i hoʻokaʻawale ʻia i ʻumi kaukani o ka PCR reactions, ka mea i puʻunaue ʻia i nā chips a i ʻole microdroplets, i loaʻa i kēlā me kēia pane ka nui o kahi mole template, a hoʻokō ʻia kahi hopena PCR hoʻokahi-mole.Ma ka heluhelu ʻana i ka fluorescence Ua helu ʻia ka hele ʻana a i ʻole ka haʻalele ʻana o ka hōʻailona, a hana ʻia ka helu piha ma hope o ka calibration o ka puʻunaue Poisson helu.
Eia nā hiʻohiʻona o kekahi mau pūnaewele PCR kikohoʻe aʻu i hoʻohana ai:
1. Bio-Rad QX200 droplet kikohoʻe PCR Bio-RadʻO QX200 kahi kahua PCR kikohoʻe maʻamau, ʻo ke kaʻina hana ʻike maʻamau: 20,000 mau laʻana i hana ʻia e ka mea hoʻoheheʻe droplet Ua hoʻonui ʻia nā micro-droplets wai-in-oil ma kahi mīkini PCR maʻamau, a ʻo ka hope o ka hōʻailona fluorescence o kēlā me kēia micro-droplet e heluhelu ʻia e ka mea heluhelu micro-droplet.ʻOi aku ka paʻakikī o ka hana, a ʻoi aku ka nui o ka pollution.
Xinyi TD1 micro-droplet digital PCRʻO Xinyi TD1 kahi kahua PCR kikohoʻe kūloko, ke kaʻina hana ʻike maʻamau: e hoʻopuka i 30,000-50,000 wai i loko o ka ʻaila droplets ma o ka droplet generator, hoʻonui i kahi mea hana PCR maʻamau, a hala hope.Hoʻokō ʻia nā hanauna droplet ʻelua a me ka heluhelu ʻana ma kēia kahua i loko o kahi pahu i hoʻolaʻa ʻia me ka haʻahaʻa haʻahaʻa o ka haumia.
STILLA Naica micro-droplet chip digital PCRʻO STILLA Naica kahi kahua PCR kikohoʻe hou.ʻO ke kaʻina hana ʻike maʻamau: e hoʻohui i ka hopena hopena i ka chip, e hoʻokomo i ka chip i loko o ka micro-droplet generation a me ka ʻōnaehana hoʻonui, a hoʻohua i 30,000 micro-droplets.Laha ma ka chip, a hoʻopau ʻia ka PCR amplification ma ka chip.A laila, hoʻololi ʻia ka chip i hoʻonui ʻia i ka ʻōnaehana heluhelu heluhelu micro-droplet, a heluhelu ʻia ka hōʻailona fluorescent ma ke kiʻi ʻana i nā kiʻi.Ma muli o ke kaʻina hana holoʻokoʻa i loko o kahi puʻupuʻu i pani ʻia, ua haʻahaʻa ka pilikia o ka haumia.
4. ThermoFisher QuantStudio 3D chip kikohoʻe PCR
ʻO ThermoFisher QuantStudio 3D kahi kahua PCR kikohoʻe maʻamau.ʻO kāna kaʻina hana ʻike maʻamau: e hoʻohui i ka hopena hopena i loko o ka mea hoʻolaha, a hoʻolaha i ka hopena hopena ma luna o ka chip me 20,000 microwells ma o ka mea hoʻolaha., kau i ka chip ma ka mīkini PCR e hoʻonui ai, a hope loa e hoʻokomo i ka chip i loko o ka mea heluhelu a lawe i ke kiʻi e heluhelu ai i ka hōʻailona fluorescent.He paʻakikī ka hana, a ua hana ʻia ke kaʻina holoʻokoʻa i loko o kahi pahu paʻa, a haʻahaʻa ka pilikia o ka contamination.
5. JN MEDSYS Clarity chip kikohoʻe PCR
ʻO JN MEDSYS Clarity kahi kahua PCR kikohoʻe hou.ʻO kāna kaʻina hana ʻike kumu: e hoʻohui i ka hopena hopena i loko o ka applicator, a hoʻolaha like i ka hopena hopena ma nā paipu PCR 10,000 i hoʻopaʻa ʻia i ka paipu PCR ma o ka applicator.Ma ka microporous chip, komo ka hopena hopena i ka chip ma o ka hana capillary, a kau ʻia ka paipu PCR me ka chip ma luna o ka mīkini PCR no ka hoʻonui ʻana, a hope loa e hoʻokomo ʻia ka chip i ka mea heluhelu e heluhelu i ka hōʻailona fluorescent ma ke kiʻi ʻana i ke kiʻi.ʻOi aku ka paʻakikī o ka hana.He haʻahaʻa ka pilikia o ka haumia.
Ua hōʻuluʻulu ʻia nā ʻāpana o kēlā me kēia kahua PCR kikohoʻe penei:
ʻO nā hōʻailona loiloi o ka pānaʻi PCR kikohoʻe: ka helu o nā ʻāpana ʻāpana, ka helu o nā kahawai fluorescent, ka paʻakikī o ka hana a me ka pilikia o ka haumia.Akā ʻo ka mea nui loa ka ʻike pololei.ʻO kahi ala e loiloi ai i nā paepae PCR kikohoʻe, ʻo ia ka hoʻohana ʻana i nā paepae PCR kikohoʻe lehulehu e hōʻoia i kekahi i kekahi, a ʻo kekahi ala ʻē aʻe, ʻo ia ka hoʻohana ʻana i nā mea maʻamau me nā waiwai pololei.
Nā pōmaikaʻi o ka dPCR
1.Loaʻa i ka quantification piha
2.ʻOi aku ka naʻau a me ka kikoʻī
3.Hiki ke ʻike i nā laʻana kope haʻahaʻa
Nā hemahema o ka dPCR1. ʻO nā lako a me nā reagents makamae 2. ʻO ka hana paʻakikī a me ka manawa ʻike lōʻihi.
I kēia manawa, loaʻa i nā hanauna ʻekolu o ka ʻenehana PCR ko lākou pono ponoʻī a me nā pōʻino, a loaʻa i kēlā me kēia me kāna mau noi ponoʻī, ʻaʻole ia he pilina e pani ai kekahi hanauna i kekahi.ʻO ka holomua mau o ka ʻenehana ua hoʻokomo i ka ikaika hou i loko o ka ʻenehana PCR, hiki iā ia ke wehe i kahi kuhikuhi noi ma hope o kekahi, e ʻoi aku ka maʻalahi a me ka pololei o ka ʻike nucleic acid.
Puna: Lawe ʻo Kauka Yuan iā ʻoe no ka hoʻāʻo
Manaʻo ʻia nā huahana:
Ka manawa hoʻouna: Nov-18-2022
