Dab tsi yog DNA hybridization? Txawm hais tias ob-stranded DNA sib lawv liag feem ntau ruaj khov nyob rau hauv physiological tej yam kev mob, hloov tej yam kev mob nyob rau hauv lub laboratory (feem ntau yog los ntawm tsa lub ambient kub) yuav ua rau cov molecules cais mus rau hauv ib tug neeg strands. Cov tom kawg yog ua tiav rau ib leeg, tab sis kuj tseem tuaj yeem ua tiav lwm cov kab ke tam sim no hauv lawv ib puag ncig. Txo qhov kub ntawm qhov chaw tso cai rau ib leeg-stranded molecules rau anneal lossis "hybridize" nrog ib leeg. Nov yog txoj kev DNA hybridization.
Lub tswvyim los ntawm qhov kev xav ntawm molecular biology
Cov kws tshawb fawb koom nrog hauv ob qho tib si DNA replication thiab kev hloov pauv ntawm DNA rau hauv RNA vam khom ntawm nucleotide crossovers thiab molecular biology cov tswv yim. Qhov no suav nrog Southern thiab Northern blots, polymerase chain reaction (PCR), thiab feem ntau DNA-RNA hybridization thiab sequencing mus kom ze.
Daim ntawv thov
Hybridization yog cov cuab yeej tseem ceeb ntawm nucleotidesequences thiab yog siv nyob rau hauv ntau txoj kev ntawm molecular biology. Tag nrho cov caj ces kev sib raug zoo ntawm ob hom tuaj yeem txiav txim siab los ntawm hybridizing ntu ntawm lawv cov DNA (DNA-DNA hybridization). Vim muaj qhov sib xws ntawm cov kab mob sib xws, qhov kub siab dua yuav tsum tau yaj cov DNA hybrids piv rau cov kab mob nyob deb. Ntau txoj hauv kev siv hybridization los txiav txim qhov keeb kwm ntawm DNA qauv, suav nrog cov tshuaj tiv thaiv polymerase saw (PCR). Hauv lwm txoj kev, cov DNA luv luv yog hybridized rau cellular mRNA txhawm rau txheeb xyuas cov noob caj noob ces. Cov tuam txhab lag luam tab tom tshawb nrhiav kev siv tshuaj tiv thaiv RNA los khi rau mRNA tsis xav tau, tiv thaiv ribosome los ntawm kev txhais mRNA rau hauv cov protein.
DNA-DNA hybridization feem ntau yog hais txog cov txheej txheem molecular biology uas ntsuas cov qib ntawm caj ces zoo sib xws ntawm cov pas dej ntawm DNA sib lawv liag. Nws yog feem ntau siv los txiav txim qhov kev ncua deb ntawm ob kab mob. Nws tau siv dav hauv phylogeny thiab taxonomy.
Methodology
DNA los ntawm ib kab mob tau sau npe, tom qab ntawd sib xyaw nrog DNA tsis muaj npe uas tuaj yeem muab piv rau nws. Qhov sib tov yog incubated kom cia cov DNA strands dissociate thiab ces txias los tsim ib tug regenerated hybrid ob stranded DNA. Hybridized sequences nrog ib tug high degree ntawm zoo sib xws yuav khi ntau nruj thiab yuav tsum tau ntau zog los cais lawv: piv txwv li, lawv cais thaum rhuab ntawm ib tug siab dua. Qhov kub thiab txias dua li qhov sib txawv, cov txheej txheem hu ua "DNA melting".
YDNA melting
Kev ntsuas qhov melting profile ntawm hybridized DNA, ob-stranded DNA yog khi rau ib tug thiaj li hu ua "kem" thiab cov resulting sib tov yog rhuab. Ntawm txhua kauj ruam, kem raug ntxuav thiab DNA cov kab ke uas yaj ua ib txoj hlua khi thiab ntxuav tawm ntawm kem. Qhov kub ntawm cov ntawv sau DNA tawm hauv kab qhia txog qhov zoo sib xws ntawm cov kab ke (thiab tus qauv folding nws tus kheej ua haujlwm tswj). Cov txiaj ntsig no tau ua ke los txiav txim siab txog qib ntawm caj ces zoo sib xws ntawm cov kab mob. Raws li niaj hnub microbiology, DNA hybridization yog tsis yooj yim sua yam tsis to taub tej yam no.
Thaum ntau hom ribonucleic acid (los yog deoxyribonucleic) cov kua qaub raug muab piv rau hauv txoj kev no, cov txiaj ntsig zoo sib xws tso cai rau cov tsiaj tso rau hauv tsob ntoo phylogenetic. Yog li ntawd, qhov no yog ib qho ntawm txoj hauv kev los ua cov txheej txheem molecular. Charles Sibley thiab John Ahlquist, cov pioneers ntawm cov txheej txheem no, siv DNA-DNA hybridization los kawm txog kev sib raug zoo ntawm cov noog (Sibley-Ahlquist taxonomy) thiab primates.
Qhov tseem ceeb rau biology
DNA-DNA hybridization yog tus qauv kub rau kev paub qhov txawv ntawm cov kab mob, nrog rau qhov zoo sib xws ntawm ntau dua 70%, qhia tias cov kab sib piv yog sib txawv. Hauv xyoo 2014, qhov pib ntawm 79% zoo sib xws tau npaj rau kev cais cov kab mob subspecies.
Cov neeg thuam hais tias cov txheej txheem tsis raug rau kev sib piv cov tsiaj sib raug zoo, raws li kev sim ntsuas qhov sib txawv ntawm qhov sib txawv orthologous ntawm cov kab mob yog dhau los ntawm hybridization ntawm paralogous counterparts hauv kab mob genome. DNA sequencing thiab computational sequence comparisons yog tam sim no feem ntau siv txoj kev los txiav txim siab nyob deb ntawm caj ces, txawm hais tias txoj kev no tseem siv nyob rau hauv microbiology los pab txheeb xyuas cov kab mob.
Txoj kev tam sim no yog ua DNA-DNA hybridization hauv silicone siv tag nrho lossis ib feem ntawm cov genomes. GGDC tsim los ntawm DSMZ yog cov cuab yeej paub tseeb tshaj plaws rau kev suav cov txiaj ntsig zoo li DDH. Ntawm lwm qhov kev txhim kho algorithmic, nws daws qhov teeb meem nrog paralogous sequences los ntawm ua tib zoo lim lawv tawm ntawm qhov sib tw ntawm ob genome sequences.
FISH method
Fluorescence In Situ Hybridization (FISH) yog cov txheej txheem kuaj siv los kuaj xyuas thiab ua raws DNA, feem ntau ntawm ib qho chromosome.
Nyob rau xyoo 1969, Joseph Gall thiab Mary Lou Pardu tau luam tawm ib daim ntawv qhia tias cov ntawv xov tooj cua ntawm cov ribosomal DNA ib ntus tuaj yeem siv los txheeb xyuas cov DNA sib txuas ntxiv hauv lub nucleus ntawm Qav qe. Txij li cov kev soj ntsuam thawj zaug no, ntau qhov kev kho kom zoo tau nce ntau yam thiablub rhiab heev ntawm cov txheej txheem rau xws li ib tug raws li nyob rau hauv situ hybridization ("nyob rau hauv qhov chaw", Latin) yog tam sim no suav hais tias yog ib qho tseem ceeb cuab tam nyob rau hauv cytogenetics. (Lub sij hawm nyob rau hauv situ tam sim no kuj tau siv los hais txog thawj theem ntawm kev loj hlob ntawm carcinoma, thaum tsuas yog cov ntaub so ntswg epithelial koom nrog cov txheej txheem pathological.)
Fluorescent hybridization sequence
RNA kev sojntsuam tuaj yeem tsim los rau txhua tus noob lossis ib ntu hauv cov noob kom pom lncRNA thiab miRNA mRNA hauv cov ntaub so ntswg thiab hlwb. FISH yog siv los ntawm kev kawm txog lub voj voog ntawm kev tsim tawm ntawm tes, tshwj xeeb tshaj yog nuclear interphase rau ib qho kev txawv txav ntawm chromosomal. FISH tso cai rau koj los tshuaj xyuas cov xwm txheej loj ntawm cov ntaub ntawv khaws tseg, nws yooj yim dua los txheeb xyuas cov chromosome uas tau txheeb xyuas los ntawm kev tsim cov kev sojntsuam nrog lub hauv paus chromosome uas yuav nyiam cov chromosomes zoo sib xws.
Hybridization signals rau txhua qhov kev sojntsuam thaum kuaj pom qhov txawv txav ntawm lub cev: txhua mRNA thiab lncRNA kuaj xyuas muaj 20 khub ntawm oligonucleotides, txhua khub npog qhov chaw ntawm 40-50 bp. p. Kev sojntsuam siv cov tshuaj chemistry los kuaj mRNA.
YHybridization nrog DNA sojntsuam
Kev sojntsuam feem ntau yog tsim los ntawm DNA tawg uas tau muab cais tawm, ua kom huv thiab nthuav dav rau kev siv hauv kev tsim ntawm tib neeg genome. Qhov loj ntawm tib neeg genome yog qhov loj heev piv rau qhov ntev uas tuaj yeem ncaj qha ua raws li qhov yuav tsum tau muab faib uatawg tawg. Thaum kawg, cov fragments no tau txiav txim los ntawm kev zom ib daim qauv ntawm txhua qhov seem mus rau hauv cov chav me me uas siv cov kab ke tshwj xeeb endonucleases los ntsuas qhov loj ntawm txhua qhov me me uas siv qhov loj me me tsis suav nrog chromatography siv cov ntaub ntawv no los txiav txim seb qhov twg loj fragments overlapped nrog ib leeg..
Txhawm rau khaws cov ntsiab lus nrog lawv tus kheej DNA ib ntus, cov khoom tawg tau ntxiv rau cov kab ke ntawm cov kab mob rov ua dua tshiab. Clonal pejxeem ntawm cov kab mob, txhua tus pej xeem tuav ib leeg chromosome, tau muab khaws cia rau hauv ntau lub chaw kuaj mob thoob ntiaj teb. Artificial chromosomes (BACs) tuaj yeem loj hlob, muab rho tawm thiab sau npe rau hauv ib qho chaw kuaj mob uas muaj lub tsev qiv ntawv. Cov tsev qiv ntawv genomic feem ntau muaj npe tom qab cov tsev kawm uas lawv tau tsim. Ib qho piv txwv yog lub tsev qiv ntawv RPCI-11, muaj npe tom qab Roswell Cancer Institute hauv Buffalo (New York, USA). Cov fragments no ua txog 100 txhiab lub hauv paus khub thiab yog lub hauv paus ntawm feem ntau FISH probes.
