Cov kab mob genomic ntawm cov kab mob feem ntau tau paub.Nucleic acid probes uas yog luv luv ntawm DNA tsim los hybridize nrog ntxiv cov kab mob DNA lossis RNA ntu.Cov tshuaj tiv thaiv kab mob polymerase (PCR) yog cov txheej txheem zoo dua rau kev kuaj kab mob.Cov txheej txheem kuaj mob siab dhau tau tsim tawm tsis ntev los no.
A. Nucleic acid hybridization txheej txheem
Nucleic acid hybridization, feem ntau suav nrog Southern blotting (Southern) thiab Northern blotting (Northern), yog ib qho kev txhim kho sai sai hauv kev kuaj kab mob.Qhov laj thawj ntawm qhov kev ntsuam xyuas hybridization yog siv cov ntu luv ntawm DNA (hu ua "kev sojntsuam") tsim los ua kom sib xyaw nrog cov kab mob DNA sib txuas lossis RNA ntu.Los ntawm cua sov los yog alkaline kev kho mob, ob-stranded lub hom phiaj DNA los yog RNA raug muab cais ua ib tug strands thiab ces yog immobilized ntawm ib tug khoom txhawb nqa.Tom qab ntawd, kev sojntsuam yog ntxiv thiab hybridized nrog lub hom phiaj DNA lossis RNA.Raws li kev sojntsuam tau sau nrog isotope lossis tsis yog-radioactive nuclide, lub hom phiaj DNA lossis RNA tuaj yeem kuaj pom los ntawm autoradiography lossis los ntawm biotin-avidin system.Txij li feem ntau ntawm cov kab mob kis tau raug cloned thiab ua ntu zus, lawv tuaj yeem tshawb pom siv cov kab mob tshwj xeeb raws li kev soj ntsuam hauv cov qauv.Tam sim no, cov txheej txheem hybridization suav nrog: dot blot , in situ hybridization hauv hlwb , DNA blotting (DNA) (Southern blot) thiab RNA blotting (RNA) (Northern blot).
B.PCR Technology
Nyob rau hauv xyoo tas los no, ib tug series ntawm in vitro nucleic acid amplification cov tswv yim tau tsim raws li PCR, mus kuaj insensitive los yog uncultivable kab mob.PCR yog ib txoj hauv kev uas tuaj yeem tsim cov DNA ib ntus los ntawm cov tshuaj tiv thaiv vitro polymerase.Cov txheej txheem ntawm PCR suav nrog lub voj voog thermal ntawm peb kauj ruam: denaturation , annealing , thiab txuas ntxiv Thaum kub siab (93 ℃ ~ 95 ℃), ob txoj hlua DNA tau muab cais ua ob leeg DNA strands;tom qab ntawd ntawm qhov kub thiab txias (37 ℃ ~ 60 ℃), ob qhov sib xyaw ua ke nucleotide primers anneal rau cov ntu DNA ntxiv;whereas ntawm qhov tsim nyog kub rau Taq enzyme (72 ℃), synthesis ntawm DNA tshiab chains pib los ntawm primer 3'end siv complementary DNA raws li templates thiab ib tug nucleotides raws li cov ntaub ntawv.Yog li tom qab txhua lub voj voog, ib txoj hlua DNA tuaj yeem ua kom zoo rau hauv ob txoj hlua.Rov ua cov txheej txheem no, txhua DNA cov saw hlau sib txuas hauv ib lub voj voog tuaj yeem siv los ua qauv hauv lub voj voog tom ntej, thiab cov DNA chains yog ob npaug rau txhua lub voj voog, uas txhais tau hais tias cov khoom ntawm PCR yog amplified nyob rau hauv 2n cav ceev.Tom qab 25 mus rau 30 lub voj voog, kev tsim cov PCR raug txheeb xyuas los ntawm electrophoresis, thiab cov khoom DNA tshwj xeeb tuaj yeem pom hauv UV teeb (254nm).Rau nws qhov kom zoo dua ntawm qhov tshwj xeeb, rhiab heev, thiab yooj yim, PCR tau txais kev pom zoo hauv kev kuaj mob ntawm ntau tus kab mob xws li HCV, HIV, CMV, thiab HPV.Raws li PCR rhiab heev, nws tuaj yeem ntes tus kab mob DNA ntawm qib fg, kev ua haujlwm yuav tsum ua tib zoo ua kom tsis txhob muaj qhov tsis zoo.Tsis tas li ntawd, qhov txiaj ntsig zoo hauv kev kuaj nucleic acid tsis txhais tau tias muaj tus kab mob nyob hauv cov qauv.
Nrog rau kev siv dav ntawm cov txheej txheem PCR, cov txheej txheem tshiab thiab cov txheej txheem yog tsim los ntawm cov txheej txheem PCR rau lub hom phiaj sib txawv.Piv txwv li, lub sij hawm tiag tiag kom muaj nuj nqis PCR tuaj yeem ntes cov kab mob kis tau;nyob rau hauv situ PCR yog siv los txheeb xyuas tus kab mob nyob rau hauv cov ntaub so ntswg los yog hlwb;PCR nested tuaj yeem nce qhov tshwj xeeb ntawm PCR.Ntawm lawv, PCR lub sijhawm tiag tiag tau tsim kho sai dua.Ntau cov tswv yim tshiab, xws li TaqMan hydrolysis sojntsuam, hybridization sojntsuam, thiab molecular beacon sojntsuam, tau muab tso ua ke rau hauv lub sij hawm ntau npaum li cas PCR txheej txheem, uas yog dav siv nyob rau hauv kev tshawb fawb soj ntsuam.Dhau li ntawm kev txheeb xyuas cov kab mob hauv cov neeg mob lub cev kua dej kom raug, txoj kev no kuj tuaj yeem siv los txheeb xyuas cov tshuaj tiv thaiv kab mob.Yog li ntawd, PCR ntawm lub sijhawm tiag tiag yog siv rau hauv kev tshuaj xyuas cov txiaj ntsig zoo thiab kev saib xyuas tshuaj tiv thaiv.
C. High-throughput nrhiav tau tus kab mob nucleic acids
Txhawm rau ua kom tau raws li qhov xav tau rau kev kuaj mob sai sai ntawm cov kab mob sib kis tshiab, ntau txoj hauv kev tshawb pom tau zoo, zoo li DNA chips (DNA), tau tsim.Rau DNA chips, cov kev tshwm sim tshwj xeeb yog synthesized thiab txuas rau cov dens me me kom tsim cov DNA microarray (DNA) uas tuaj yeem yog cov qauv.Lub teeb liab ntawm hybridization tuaj yeem ua duab los ntawm confocal microscope lossis laser scanner thiab txuas ntxiv ua tiav los ntawm lub khoos phis tawj thiab cov ntaub ntawv loj loj hais txog cov noob sib txawv tuaj yeem tau txais.Muaj ob hom DNA nti.Cov "synthesis nti" yog raws li nram no: cov oligonucleotides tshwj xeeb yog synthesized ncaj qha rau ntawm daim tawv nqaij.Lwm qhov yog DNA pool chip.Cov noob caj noob ces lossis PCR cov khoom lag luam raug txiav txim siab luam tawm ntawm tus swb.Qhov kom zoo dua ntawm DNA chip thev naus laus zis yog kev tshawb nrhiav ib txhij ntawm ntau qhov DNA sib lawv liag.Qhov tseeb version ntawm cov kab mob tshawb nrhiav nti tuaj yeem txheeb xyuas ntau dua 1700 tus kab mob tib neeg ib zaug.DNA nti thev naus laus zis daws cov teeb meem ntawm cov qauv nucleic acid hybridization txoj kev thiab muaj kev siv dav dav hauv kev kuaj kab mob thiab kev tshawb fawb txog kev kis mob.
Post lub sij hawm: Dec-23-2020