Ntau qhov kev hloov pauv hloov pauv hauv keeb kwm ntawm kev tshawb nrhiav thev naus laus zis hauv kuv lub siab yog immunolabeling thev naus laus zis raws li lub hauv paus ntsiab lus ntawm antigen-antibody tshwj xeeb khi, PCR technology thiab sequencing technology.Niaj hnub no peb yuav tham txog PCR technology.Raws li kev hloov pauv ntawm PCR thev naus laus zis, tib neeg ib txwm faib PCR thev naus laus zis ua peb tiam: PCR thev naus laus zis zoo ib yam, lub sijhawm siv hluav taws xob ntau PCR thev naus laus zis thiab digital PCR thev naus laus zis.
Common PCR txheej txheem
KARY MULLIS (1944.12.28-2019.8.7)
Kary Mullis tau tsim cov tshuaj tiv thaiv polymerase saw (polymerase saw cov tshuaj tiv thaiv, PCR) hauv xyoo 1983. Nws tau hais tias thaum nws tsav tsheb nws tus hluas nkauj, nws mam li nco dheev muaj kev tshoov siab thiab xav txog lub hauv paus ntsiab lus ntawm PCR (ntawm cov txiaj ntsig ntawm kev tsav tsheb).Kary Mullis tau txais txiaj ntsig Nobel nqi zog hauv Chemistry hauv 1993. New York Times tau hais tias: "Qhov tseem ceeb heev thiab tseem ceeb, yuav luag faib ua biology rau hauv pre-PCR thiab post-PCR eras.
Lub hauv paus ntsiab lus ntawm PCR: Raws li catalysis ntawm DNA polymerase, niam strand DNA yog siv los ua tus qauv, thiab cov primer tshwj xeeb yog siv los ua qhov pib ntawm kev txuas ntxiv, thiab tus ntxhais strand DNA ntxiv rau niam strand template DNA yog theej hauv vitro los ntawm denaturation, annealing, extension thiab lwm yam.Nws yog DNA synthesis amplification thev naus laus zis hauv vitro, uas tuaj yeem ua kom nrawm thiab tshwj xeeb tshaj tawm txhua lub hom phiaj DNA hauv vitro.
Qhov zoo ntawm PCR zoo tib yam
1.Classic txoj kev, ua kom tiav thoob ntiaj teb thiab domestic qauv
2.Tsawg tus nqi ntawm cov cuab yeej reagent
3.Cov khoom PCR tuaj yeem rov qab los rau lwm qhov kev sim tshuaj lom neeg molecular
Pom zoo Foregene PCR tshuab: https://www.foreivd.com/foreamp-sn-695-series-thermal-cycler-96-wells-pcr-machine-product/
Yam khoom: https://www.foreivd.com/pcr-herotm-with-dye-product/
Disadvantages ntawm zoo tib yam PCR
1.yooj yim ua paug
2.kev ua haujlwm nyuaj
3.tsuas yog qualitative tsom xam
4.Nruab nrab rhiab heev
5.Muaj tsis yog qhov tshwj xeeb amplification, thiab thaum lub band tsis tshwj xeeb yog tib yam li lub hom phiaj band, nws tsis tuaj yeem paub qhov txawv.
Capillary electrophoresis-raws li PCR
Hauv kev teb rau qhov tsis txaus ntawm PCR zoo tib yam, qee lub tuam txhab tau qhia cov cuab yeej raws li txoj cai ntawm capillary electrophoresis.Cov kauj ruam electrophoresis tom qab PCR amplification tiav hauv capillary.Lub rhiab heev yog siab dua, thiab qhov sib txawv ntawm ntau lub hauv paus tuaj yeem paub qhov txawv thiab qhov kev nthuav dav tuaj yeem suav los ntawm MAERKER.cov ntsiab lus ntawm cov khoom.Qhov tsis zoo yog tias cov khoom PCR tseem yuav tsum tau qhib thiab muab tso rau hauv qhov ntsuas, thiab tseem muaj kev pheej hmoo loj ntawm kev sib kis.
CapillaryEelectrophoresis
2. Real-time fluorescent quantitative PCR (Quantitative Real-time PCR, qPCR) technologyFluorescent quantitative PCR, tseem hu ua Real-Time PCR, yog ib tug tshiab nucleic acid quantitative technology tsim los ntawm PE (Perkin Elmer) nyob rau hauv 1995. Cov keeb kwm kev loj hlob ntawm fluorescent quantitative PCR yog keeb kwm ntawm ntsuj plig-stirring tawm tsam ntawm giants xws li ABI, Roche, thiab Bio-Rad.Yog tias koj txaus siab, koj tuaj yeem tshawb xyuas nws.Cov txheej txheem no yog tam sim no paub tab thiab siv dav siv cov txheej txheem PCR semi-quantitative.
Pom zoo qPCR Tshuab:https://www.foreivd.com/mini-real-time-pcr-system-forequant-sf2sf4-product/
Fluorescent dye method (SYBR Green I):SYBR Green I yog qhov feem ntau siv DNA-binding dye rau ntau PCR, uas khi tsis yog tshwj xeeb rau ob-stranded DNA.Nyob rau hauv lub xeev dawb, SYBR Green emits tsis muaj zog fluorescence, tab sis ib zaug khi rau ob-stranded DNA, nws fluorescence nce 1000-fold.Yog li ntawd, tag nrho cov teeb liab fluorescent emissions los ntawm cov tshuaj tiv thaiv yog proportional rau tus nqi ntawm ob-stranded DNA tam sim no thiab yuav nce ntxiv nrog rau cov khoom amplified.Txij li cov dyes khi tsis yog tshwj xeeb rau ob-stranded DNA, cov txiaj ntsig tsis zoo yuav raug tsim tawm.
Yam khoom: https://www.foreivd.com/real-time-pcr-easytm-sybr-green-i-kit-product/
Fluorescent probe method (Taqman technology): Thaum lub sij hawmPCR amplification, ib qho kev sojntsuam fluorescent tshwj xeeb yog ntxiv rau tib lub sijhawm raws li ib khub ntawm primers.Qhov kev sojntsuam yog ib txoj kab oligonucleotide, nrog rau pawg neeg tshaj xov xwm fluorescent thiab pawg fluorescent quencher raws li sau rau ntawm ob qho kawg.Thaum qhov kev sojntsuam tsis zoo, lub teeb liab fluorescent emitted los ntawm pawg neeg sau xov xwm yog absorbed los ntawm pawg quencher, thiab qhov kev kuaj pom tsis muaj fluorescent teeb liab;Thaum lub sij hawm PCR amplification (nyob rau hauv lub ncua sij hawm), lub 5'-3' Dicer kev ua ntawm Taq enzyme yuav zom thiab degrade lub sojntsuam, thiaj li hais tias cov reporter fluorescent pab pawg neeg thiab quencher fluorescent pawg sib cais, thiaj li hais tias lub fluorescence xyuas system lub fluorescent teeb liab yuav tau txais, uas yog, txhua txhua lub sij hawm ib tug DNA saw yog amplified, ib daim ntawv ntawm cov fluorescent synchronization. Cov teeb liab fluorescent thiab tsim cov khoom PCR.Taqman sojntsuam txoj kev yog txoj kev tshawb nrhiav feem ntau siv hauv kev kuaj mob.
Yam khoom: https://www.foreivd.com/quickeasy%e1%b5%80%e1%b4%b9-real-time-pcr-kit-taqman-product/
Qhov zoo ntawm qPCR
1.Cov txheej txheem yog mature thiab cov khoom txhawb nqa thiab reagents tiav
2.Tus nqi nruab nrab ntawm reagents
3.yooj yim siv
4.High detection rhiab heev thiab tshwj xeeb
Disadvantages ntawm qPCR
Kev hloov pauv ntawm lub hom phiaj noob ua rau tsis pom kev.
Qhov kev kuaj pom ntawm cov qauv qis qis tsis tuaj yeem txiav txim siab.
Muaj qhov yuam kev loj thaum siv tus qauv nkhaus rau kev tshawb nrhiav kom muaj nuj nqis.
3. Digital PCR (digital PCR, dPCR) tshuab
Digital PCR yog cov txheej txheem rau kev ua kom muaj nuj nqis ntawm nucleic acid molecules.Piv nrog rau qPCR, digital PCR tuaj yeem ncaj qha nyeem cov lej ntawm DNA / RNA molecules, uas yog qhov muaj tseeb ntawm nucleic acid molecules hauv cov qauv pib.Xyoo 1999, Bert Vogelstein thiab Kenneth W. Kin-zler tau tsim lub tswv yim ntawm dPCR.
Xyoo 2006, Fluidigm yog thawj tus tsim cov khoom lag luam nti-raws li dPCR ntsuas.Hauv xyoo 2009, Lub Neej Technologies tau tshaj tawm OpenArray thiab QuantStudio 12K Flex dPCR systems.Hauv xyoo 2013, Lub Neej Technologies tau tshaj tawm QuantStudio 3DdPCR system, uas siv cov tshuab siv high-density nanoscale microfluidic nti kom sib npaug faib cov qauv mus rau 20,000 tus neeg lub hlwb.nyob rau hauv cov tshuaj tiv thaiv zoo.
Nyob rau hauv 2011, Bio-Rad tau tsim lub tee-raws li QX100 dPCR ntsuas, uas siv dej-hauv-roj thev naus laus zis los faib cov qauv mus rau 20,000 tee dej-dej-hauv-roj, thiab siv lub ntsuas dej los ntsuas cov tee dej.Hauv xyoo 2012, RainDance tau tsim cov cuab yeej RainDrop dPCR, uas tau tsav los ntawm cov pa roj siab, los faib cov txheej txheem tshuaj tiv thaiv rau hauv cov tshuaj tiv thaiv emulsion uas muaj 1 lab rau 10 lab picoliter-theem micro-droplets.
Txog tam sim no, cov PCR digital tau tsim ob pawg loj, hom nti thiab hom droplet.Tsis muaj teeb meem dab tsi ntawm PCR digital, nws cov ntsiab lus tseem ceeb yog txwv tsis pub muaj kev cuam tshuam, qhov kawg ntawm PCR thiab Poisson faib.Tus txheej txheem PCR cov tshuaj tiv thaiv uas muaj cov qauv nucleic acid yog sib npaug rau kaum tawm txhiab tus PCR cov tshuaj tiv thaiv, uas tau muab faib rau cov chips lossis microdroplets, kom txhua qhov tshuaj tiv thaiv muaj ntau npaum li qhov ua tau ntawm cov qauv qauv, thiab ib qho qauv qauv PCR cov tshuaj tiv thaiv yog ua.Los ntawm kev nyeem cov fluorescence Lub xub ntiag los yog tsis muaj lub teeb liab raug suav, thiab qhov tseeb kom muaj nuj nqis yog ua tom qab calibration ntawm cov txheeb cais Poisson faib.
Cov hauv qab no yog cov yam ntxwv ntawm ntau lub PCR digital platforms kuv tau siv:
1. Bio-Rad QX200 droplet digital PCR Bio-RadQX200 yog qhov zoo tshaj plaws digital PCR platform, cov txheej txheem tshawb nrhiav yooj yim: 20,000 cov qauv yog tsim los ntawm lub tshuab hluav taws xob cov dej hauv cov roj micro-tej dej yog nthuav dav ntawm lub tshuab PCR zoo tib yam, thiab thaum kawg cov teeb liab fluorescence ntawm txhua lub micro-droplet nyeem los ntawm micro-droplet nyeem ntawv.Kev ua haujlwm yog qhov nyuaj dua, thiab qhov kev pheej hmoo ntawm kev ua qias tuaj yog nruab nrab.
Xinyi TD1 micro-droplet digital PCRXinyi TD1 yog lub hauv paus digital PCR platform, cov txheej txheem tshawb nrhiav yooj yim: tsim 30,000-50,000 dej-hauv-roj cov tee dej los ntawm lub tshuab hluav taws xob tee, ua kom nrov nrov ntawm cov cuab yeej PCR, thiab thaum kawg dhau Tus neeg nyeem ntawv nyeem cov fluorescent teeb liab ntawm txhua lub tee dej.Ob lub cim xeeb thiab kev nyeem ntawv hauv lub platform no tau ua nyob rau hauv lub siab tawv nti uas tsis tshua muaj kev pheej hmoo ntawm kev kis kab mob.
STILLA Naica micro-droplet nti digital PCRSTILLA Naica yog ib qho tshiab digital PCR platform.Cov txheej txheem tshawb nrhiav yooj yim yog: ntxiv cov tshuaj tiv thaiv rau cov nti, muab cov nti tso rau hauv lub micro-droplet tiam thiab amplification system, thiab tsim 30,000 micro-droplets.Tshaj tawm ntawm lub nti, thiab PCR amplification tiav ntawm lub nti.Tom qab ntawd lub nti amplified yog pauv mus rau micro-droplet nyeem ntawv txheeb xyuas qhov system, thiab cov teeb liab fluorescent nyeem los ntawm kev thaij duab.Txij li thaum tag nrho cov txheej txheem yuav siv qhov chaw nyob rau hauv ib tug kaw nti, qhov kev pheej hmoo ntawm kev kis kab mob yog tsawg.
4. ThermoFisher QuantStudio 3D nti digital PCR
ThermoFisher QuantStudio 3D yog lwm lub chip-based digital PCR platform.Nws cov txheej txheem tshawb nrhiav yooj yim yog: ntxiv cov tshuaj tiv thaiv rau hauv cov khoom sib kis, thiab kis tau cov tshuaj tiv thaiv sib npaug ntawm cov nti nrog 20,000 microwells los ntawm cov kis., muab cov nti tso rau ntawm lub tshuab PCR kom nthuav dav, thiab thaum kawg muab cov nti tso rau hauv tus nyeem ntawv thiab thaij duab los nyeem cov teeb liab fluorescent.Kev ua haujlwm yog qhov nyuaj, thiab tag nrho cov txheej txheem yog nqa tawm nyob rau hauv ib tug kaw nti, thiab kev pheej hmoo ntawm kev kis kab mob yog tsawg.
5. JN MEDSYS Clarity nti digital PCR
JN MEDSYS Clarity yog ib qho tshiab nti-hom digital PCR platform.Nws cov txheej txheem tshawb pom yooj yim yog: ntxiv cov tshuaj tiv thaiv rau hauv tus neeg thov, thiab kis cov tshuaj tiv thaiv sib npaug ntawm 10,000 PCR hlab tsau hauv PCR raj los ntawm tus neeg thov.Ntawm lub microporous nti, cov tshuaj tiv thaiv nkag mus rau hauv nti los ntawm kev ua haujlwm capillary, thiab PCR raj nrog cov nti tau muab tso rau ntawm lub tshuab PCR rau kev nthuav dav, thiab thaum kawg cov nti tau muab tso rau hauv tus nyeem ntawv nyeem cov teeb liab fluorescent los ntawm kev thaij duab.Kev ua haujlwm yog qhov nyuaj dua.Qhov kev pheej hmoo ntawm kev kis kab mob tsawg.
Cov kev txwv ntawm txhua qhov digital PCR platform tau sau tseg raws li hauv qab no:
Cov kev ntsuas ntsuas ntawm lub digital PCR platform yog: tus naj npawb ntawm cais units, tus naj npawb ntawm cov fluorescent raws, qhov nyuaj ntawm kev ua haujlwm thiab kev pheej hmoo ntawm kev sib kis.Tab sis qhov tseem ceeb tshaj plaws yog qhov ntsuas qhov tseeb.Ib txoj hauv kev los ntsuam xyuas cov PCR platforms yog siv ntau lub platforms digital PCR los txheeb xyuas ib leeg, thiab lwm txoj hauv kev yog siv cov txheej txheem nrog cov txiaj ntsig raug.
Qhov zoo ntawm dPCR
1.Ua kom tiav qhov muaj nuj nqis
2.Siab rhiab heev thiab tshwj xeeb
3.Muaj peev xwm kuaj xyuas cov qauv qis
Disadvantages ntawm dPCR1. Cov khoom siv kim heev thiab cov tshuaj reagents 2. Kev ua haujlwm tsis yooj yim thiab lub sijhawm tshawb nrhiav ntev 3. Kev tshawb nrhiav qhov nqaim
Tam sim no, peb tiam ntawm PCR thev naus laus zis muaj lawv tus kheej qhov zoo thiab qhov tsis zoo, thiab txhua tus muaj nws tus kheej daim ntawv thov, thiab nws tsis yog kev sib raug zoo uas ib tiam hloov pauv.Kev nce qib ntawm kev siv thev naus laus zis tau txhaj tshuaj tshiab tseem ceeb rau hauv PCR thev naus laus zis, ua kom nws qhib ib daim ntawv thov kev taw qhia tom qab lwm qhov, ua rau kev tshawb nrhiav nucleic acid yooj yim dua thiab raug.
Source: Dr. Yuan coj koj mus kuaj
Cov khoom pom zoo:
Lub sij hawm xa tuaj: Nov-18-2022
