Kwayoyin kayyade bincike Hanyar

Don bincika da kuma gane bambance-bambancen karatu a cikin DNA tsarin amfani da kwayoyin kayyade Hanyar. Ga kowane DNA yankin, wanda ya duba wannan yankin na chromosome, gene ko allele, da hanyoyin sãɓawa jũna. Muhimmi kowane kwayoyin kayyade Hanyar qunshi wasu ko wasu magudi da RNA da DNA. Duk wadannan hanyoyi ne halin da wani babban wuya, da ba tare da awon yanayi ba za a iya za'ayi, da kuma ma'aikatan dole ne sosai m. Wannan aiki ne da za'ayi a dama, saukarwa.

saukarwa

Da farko, RNA ko DNA samfurori da za a samar. A nan, wani kwayoyin kayyade Hanyar za a iya amfani da su kusan duk wani material: wani digo na jini, leukocytes, fibroblasts al'adu, mucosa (kankare), har ma da gashi follicles, - DNA za a iya samu daga duk wani samfurin. Shi ya dace don amfani da wani kwayoyin kayyade hanyoyin da su daban-daban zabin da sun dogon ware DNA da aka adana daskararre. A mataki na biyu da aka sadaukar domin jari na so, niƙaƙƙun gaɓãɓuwa (karawa) na DNA, kamar yadda yana taimaka tabbatar da polymerase sarkar dauki a vitro (a vitro ba tare da hannu na rayuwa kwayoyin). A sakamakon haka, da aka zaɓa DNA gutsure na ninka wannan sarkar dauki, da kuma adadin DNA ƙaruwa a zahiri miliyoyin sau.

Mataki na uku na cikin kwayoyin kayyade bincike hanyoyin aka zaci A riɓanya DNA ƙuntatawa (wannan fragmentation, tearing ko yankan). Ƙuntatawa yi da electrophoresis a kan wani polyacrylamide ko agarose gel. Wannan kwayoyin-kayyade Hanyar nazarin DNA gutsure damar kowa ya dauki wani matsayi a cikin gel. Bayan nan, da gel aka bi da tare da ethidium bromide, iya dauri ga DNA, da sakawa a iska mai guba da ultraviolet da haske, sa'an nan shi ne mai yiwuwa a kalla luminescence rabo. Kwayoyin kayyade bincike hanyoyin su ne bambance bambancen da yawa, amma da farko matakai biyu ne ga kowa da kowa. Amma domin ya gane DNA da wani ɓaɓɓake, da gel za a iya canza launin, da kuma da yawa wasu data kasance hanyoyin.

jinsunan

A mafi kai tsaye da kuma tartsatsi hanyoyin domin ganowa mycobacteria iya hada sama kwayoyin kayyade DNA koyo hanya. Its ainihi shi ne, domin su gane scan sarkar abu na musamman da wani ɓaɓɓake daga DNA na pathogens. Kwayoyin kayyade bincike dabaru ba tukuna da mafi m hanyar gane irin cututtuka kamar tarin fuka. Amfani da polymerase sarkar dauki (PCR), za ka iya tabbata cewa ainihin DNA zai kara yawan kofe a wani sau miliyan, wato, za a karawa, kuma shi zai nuna maka da sakamakon. The ji na ƙwarai matakin ne sosai high - fiye da casa'in da biyar cikin dari, wanda shi ne babban amfani da wannan hanya.

Sauran kwayoyin-kayyade hanyoyin da bincike a kan tasiri na amfanin ƙasa mahara kofe zahiri ninki biyu, tun a cikin wannan harka Shirin zanen samfurin nuna wani takamaiman oligonucleotide jerin karu zuwa ɗari da sau shida. Ko da al'adun ganewar asali da tarin fuka na numfashi tsarin muhimmanci runtse ta ji na ƙwarai. Wannan shi ne dalilin da ya sa zamani magani dogara ne a kan kwayoyin kayyade hanyoyin da ganewar asali da tarin fuka. A bayyana Hanyar ne tasiri musamman lokacin da akayi tare da pathogens na high antigenic canzawa, sanin cewa wasu hanya ne yafi wuya - bukatar musamman na gina jiki kafofin watsa labaru da kuma raya dogon lokaci. Biochemical da kwayoyin kayyade hanyoyin sarrafa sosai daban-daban sakamako a kan sakamakon.

ganewar asali da tarin fuka

Marshall PCR ganewar asali da tarin fuka fi yin amfani da waɗanda jerin DNA da suke da takamaiman ga duk hudu daban na cutar. Don cimma wannan burin sau da yawa amfani primers cewa gane jeren NE abubuwa (NE-986, IS-6110), kamar yadda wadannan abubuwa fahince sosai migratory jinsunan Mycobacterium da tarin fuka da kuma ko da yaushe ba mahara kofe a cikin genome. Har ila yau DNA hakar za a iya za'ayi daga m al'adu da Clinical (sputum da marasa lafiya) da wani datacewa. Alal misali, akwai Boom Hanyar inda lysis buffer aka yi amfani dangane da guanidine thiocyanate da silica a matsayin m DNA. Yawan marasa lafiya cewa bambanta matalauta bacteriological kara a kowace shekara, kuma haka a cikin asibiti yi ta kafa wani mabanbanta matakin na kungiyar: kwayoyin-kayyade Hanyar karatu DNA da aka wasa muhimmiyar rawa a cikin ganewar asali.

Duk da haka, dole ne mu yarda da cewa shi ne ba tare da drawbacks. A PCR Hanyar ne amfani da sau da yawa ya zo da wata babbar yawan ƙarya-tabbatacce sakamakon, kuma dalilin shi ne ba kawai fasaha kurakurai, amma kuma siffofin da hanyar da kanta. Bugu da kari, yin amfani da wannan hanya na ganewar asali domin sanin viability na mycobacteria, wanda da aka gano, shi ne kawai zai yiwu ba. Amma wannan hasara ba mafi muhimmanci. Kwayoyin kayyade hanyoyin da PCR bincikowa da ya kunsa da hadarin samu daga mycobacterial DNA. Takardar shaida bukatun domin wannan dalilin cewa ga PCR dakunan gwaje-gwaje na musamman tsara da wuya, suka bukatar uku raba gabatarwa. PCR fasahar ne na zamani da kuma sosai hadaddun, shi na bukatar yin amfani da dace kayan aiki da kuma high-horar da ma'aikata.

bacterioscopy

Lokacin da ganewar asali sakamakon bincike dole ne a idan aka kwatanta da sauran data: asibiti jarrabawa, radiography, shafa bincike da madubin likita, amfanin gona da kuma ko mayar da martani ga wani takamaiman magani suke da muhimmanci sosai. A cikin wannan jerin, da PCR nazarin ne kawai daya daga cikin aka gyara. Gane da pathogen a farkon ganewar asali na iya zama da sauki da kuma quickest hanyoyin - bacteriological.

Akwai aka yi amfani da wani haske microscope (kala Ziehl-Neelsen) da kuma mai kyalli (canza launi fluorochromes). A amfani ne gudun shafa sakamakon. Amma ta drawback ne gaskiya dauke da Iyakantacce saboda low ji na ƙwarai. Duk da haka, wannan hanya da aka ba WHO shawarwarin a matsayin mafi tattali da kuma ƙasa don gane da tarin fuka marasa lafiya. Ganewa na mycobacteria bacteriological hanya yana Hasashen darajar da kiyasta quantitatively kwayan tukar tumbi. Fiye da m magance shi kwayoyin kayyade bincike hanyoyin da tarin fuka.

al'adu

Better ganewa na mycobacteria gane al'adu da karatu. Shuka pathological abu an yi shi a cikin kwai matsakaici: Mordovsky, Finn II, LJ, da kuma kama. Asowar na juriya da mycobacteria zuwa magunguna da kuma kai tsaye ba da shaida na da tasiri na a yawan mycobacteria kuma su mazauna a vitro, idan amfani Hanyar bincike al'adu. Don kara yawan kadaici da mycobacteria inoculation na pathological abu ne da aka gudanar a kan mahara halin wurare.

Ganawa da bukatun yawa al'adu, pathogen ciki har da Grant da ruwaye. Amfani a cikin wannan tsarin da kuma sarrafa kansa metering irin VANTES girma. Amfanin gona dole ne a gudanar a shiryawa domin har zuwa bakwai zuwa takwas makonni. By wannan lokaci da amfanin gona tare da rashin ci gaban da za a iya daukan korau. A mafi inganci hanyar gano Mycobacterium tarin fuka la'akari da nazarin halittu samfurori: bincike abu harba Guinea aladu, wanda su ne musamman mai saukin kamuwa zuwa tarin fuka.

wasu Figures

Ban sha'awa fannin nazari, wanda aka bude PCR bincikowa da ya yi nazarin M. tarin fuka - latent kamuwa da cuta. A zamani manufar tarin fuka kamuwa da shawara cewa, daga wani ɗari mutane da suke a lamba tare da M. tarin fuka, tasa'in da zai iya da kyau za a cutar, amma kawai goma daga cikinsu akwai aiki cutar da ake ci gaba. Wasu suna da tarin fuka rigakafi, da kuma saboda casa'in cikin dari na lokuta da kamuwa da cuta da ya rage latent. Gane abin koyi da shi ya taimaka da wani kwayoyin kayyade Hanyar.

Jinsi ce cewa hamsin da biyar kashi na wadanda albarkatu pathological kayan sun korau, da tamanin cikin dari na mutanen da suka kamu da tarin fuka M., amma gudãna ba tare da wani radiographic manifestations na cuta, PCR m martani samu. Yana da wani kayyade bincike Hanyar taimaka, wajen gano marasa lafiya a hadarin da PCR karatu, tare da sakamakon da ƙididdiga (bincike da madubin likita kuma al'adun) aka korau, kuma subclinical kamuwa M. tarin fuka ya ba.

zamani bincike

The Russian Federation, kuma bacteriological dakunan gwaje-gwaje amfani da wani kara Hanyar cikakkar yawa: nitrate reductase aiki na mycobacteria gwada Griess reagent. Anti-tarin fuka cibiyoyin amfani da wata hanya da damar domin sanin miyagun ƙwayoyi juriya. Wannan seeding a cikin ruwa kafofin watsa labarai, inda sarrafa kansa radiometric kuma mai kyalli lissafin kudi tsarin girma na mycobacteria. Irin wannan bincike da aka yi sauri - har zuwa makonni biyu.

A halin yanzu, sabon hanyoyin da ake da ake ci gaba: miyagun ƙwayoyi juriya na mycobacteria aka auna a genotype matakin. Nazarin daga cikin kwayoyin sunadaran da juriya da kwayoyin halittu da kuma nuna gaban a mycobacteria. Wadannan kwayoyin halittu suna hade da juriya ga wasu kwayoyi. Alal misali, kasA kwayoyin halitta, inhA, katG resistant zuwa isoniazid, rpoB gene - rifampicin 16Sp RNA kwayoyin halittu da kuma rpsL - streptomycin, emb1 - to ethambutol, gyrA - a fluoroquinolone da sauransu.

maye gurbi

A zamani ganewar asali muhimmanci karu da kwayoyin-kayyade matakin hanya domin karatu DNA da kuma yarda da wani sashe daga manyan-sikelin karatu na maye gurbi a cikin dukkan su bakan. Yanzu mun san cewa ya fi kowa maye gurbi a cikin 516, 526 da kuma 531 codons da rpoB gene, da kuma gano jure daban-daban kwayoyi. Akwai dukan kewayon hanyoyin da bugawa na mycobacteria amfani ba kawai gargajiya hanyoyin - biochemical, nazarin halittu da kuma al'adu, amma kuma yadu amfani zamani kwayoyin kayyade dabaru. Riga akwai isassun da kuma samar da daidai ganewar asali hanya domin ganewa na monogenic cututtuka. Suna dogara ne a kan DNA nazarin a daidai yankin da wani gene. Wannan shi ne yawanci hadaddun tsari, lokacin cinyewa kuma tsada, amma data cewa an bayar da kwayoyin kayyade analysis, shi ne yafi m da m fiye da data dukkan sauran ƙididdiga.

An dade an san cewa DNA bai sauya ba domin dukan rai da kwayoyin da cewa shi ne a cikin wani nucleated Kwayoyin odnakova, kuma wannan ya sa ya yiwu ya dauki bincike na da cikakken duk Kwayoyin na jiki, a wani mataki na yanayin halitta na juya. A lalace gene za a iya gano kafin bayyanar da farko bayyanar cututtuka da cikakken sikelin asibiti cutar, kazalika da a lafiya heterozygous mutane, amma ciwon maye gurbi a cikin gene. Kwayoyin kayyade hereditary cuta bincike hanyoyin ba da damar zuwa bayyana ta (kai tsaye m, DNA-bincikowa), kazalika da mu tantance fata da cutar a cikin iyali da alama loci DNA (kayyade polymorphisms), wanda aka nasaba, tare da wani lalace gene (ie, kaikaitaccen m na DNA-bincikowa). Kai tsaye ko a kaikaice - wani DNA bincikowa dogara ne a kan hanyoyin da gano mai tsananin tsare rabo daga mutum DNA.

kai tsaye hanyoyin

Direct hanyoyin da DNA ganewar asali ne a lokacin da m gene hereditary cuta da aka sani, kamar yadda da sani, da kuma iri daban-daban ta maye gurbi. Alal misali, a dace kai tsaye hanyoyin cikin wani yawan cututtuka. Wannan Huntington ta chorea (tsawo CTG-maimata da ita), phenylketonuria (R408W), cystic fibrosis (delF508, manyan maye gurbi) da kuma kamar. Babban amfani da kai tsaye Hanyar ne dukansa-mallakar bincike daidaito, da kuma babu bukatar a yi a DNA bincike na sauran iyali. Idan wani maye gurbi a cikin m gene da aka samu, shi damar daidai amince da ganewar asali na gadar hali, genotype kafiya for Sauran nauyin iyali.

Wani amfani da kai tsaye ganewar asali aka dauke su gane wani heterozygous m na sharri maye gurbi daga dangi da kuma iyaye da suka mutu daga cutar. Wannan Gaskiya ne, ga cututtuka autosomal recessive. Disadvantages na kai tsaye hanyoyin ma suna samuwa. Don amfani da su, kana bukatar ka san daidai localize da mahaukaci gene, exon-intron tsarin ta bakan da kuma ta maye gurbi. Ba duk monogenic cututtuka a yau sun samu irin wannan bayani. Informativeness kai tsaye hanyoyin ba za a dauke da cikakken, saboda daya da kuma guda gene iya samun manyan yawan pathological maye gurbi da cewa yana sa cin gaban hereditary cututtuka.

kaikaitaccen hanyoyin

Kaikaitaccen hanyoyin a DNA bincikowa da aka yi amfani a duka, a wasu lokuta, idan lalace gene ba gano, amma kawai chromosomally, ko idan line ganewar asali bai ba da sakamakon (ta faru, idan gene hadaddun kwayoyin kungiyar ko wani babban har, idan akwai mai yawa pathological maye gurbi). Kaikaitaccen hanyoyin yin fata bincike na polymorphic alamomi a allelic iyali. Alamomi samu a cikin wannan chromosomal yankin ko wuri da aka nasaba, da cutar da kuma wakiltar deletions ko insertions, ma'ana substitutions, maimata da ita, kuma su polymorphism ne saboda daban-daban adadin Kwayoyin a cikin block.

Mafi dace domin kai tsaye ba ganewar asali dauke microsatellite da minisatellite polymorphisms, wanda aka yadu rarraba a cikin mutum genome. da darajar da aka bayyana a high bayanai abun ciki, idan lalacewar da kwayoyin nesa tsakanin alama da kuma gene ba ma manyan. A karshen harka, da hakkin daidaito aka ƙaddara ga babban har da mita na recombination tsakanin polymorphic alama da kuma lalacewa. Kaikaitaccen bincike hanyoyin samar da m na farko mataki na allele mitoci na bincikar yawan nazari tsakanin marasa lafiya da kuma dako maye gurbi, na da wajibcin kayyade Yiwuwar recombination na nonequilibrium da mannewa alamomi da mutant alleles.

sauran hanyoyin da

Short segments na RNA ko DNA, kazalika da guda gene visualized karkashin microscopic binciken ba zai iya zama, saboda haka, don gano maye gurbi yake bukata ta hanyar kwayoyin kayyade ganewar asali. Akwai "Human genome Project", kazalika da sauran ci gaba a kwayoyin halittar jini ƙwarai fadada da yiwuwar ganewar asali hereditary cututtuka - biyu xabi'a da postnatal. Wadannan hanyoyin iya samar da farkon ganewa da kuma yin Hasashen poly- da monogenic cututtuka, wanda halarta a karon faruwa a} uruciyarsa. Abin baƙin ciki, saboda da fasaha damar kwayoyin kayyade nazarin ne wani lokacin bayan da da'a iyaka cewa an kafa dangane da gado, musamman a lokacin da ganewar asali ne a samartaka da kuma yara.

Tsarin da kuma na lamba chromosomal munanan sun fi na kowa Sanadin cutar da kuma ciwon daji, da kuma da yawa malformations. Chromosomal aberrations bukatar da za a gano, wanda yana da muhimmanci ga iyali shawara - don tantance hasashen, tare da haihuwa hadarin a nan gaba ciki. Chromosome bincike ne "zinariya misali" kayyade ganewar asali, amma shi yana da iyaka. Kawai hanyoyin da kwayoyin kayyade analysis iya yin fiye da, saboda akwai amfani cloning fasahar tushen kyalli tasirin iya su high ji na ƙwarai to gane da dabara chromosomal canje-canje da suke da ba zai yiwu ba a gane gargajiya cytogenetic karatu. Wadannan dabaru suna ƙara fadada mu bincike damar, a lokacin da yayi nazari, yara da raya ci gaban nakasa, shafi tunanin mutum retardation, tare da wasu hereditary cututtuka.

binciken

Yana da matukar muhimmanci ga bil'adama su ne gene sifa da aiki na ilimi, iri canzawa, da ikon gane hereditary cututtuka da cewa ya faru dangane da ci gaban da kwayoyin halittar jini. ta hanyoyin da ake da nufin nazarin cikin jigidar halittar DNA - kuma a lokacin da shi ne al'ada, da kuma lokacin da ta lalace. Shiri na deoxyribonucleic acid jeruwar nucleotides (DNA) kara, a cikin hãlãye daga samun samfurori to gane mutum, niƙaƙƙun gaɓãɓuwa. Kadaici da ilimin kwayoyin hali DNA daga Kwayoyin, ƙuntatawa (tearing), karawa (cloning), electrophoresis na da wani ɓaɓɓake (raba su wutar lantarki da kuma kwayoyin nauyi da agarose gel). Identification na musamman, niƙaƙƙun gaɓãɓuwa located a farfajiya na wani mai hankali stripe.

Sa'an nan da samun a cikin yi musamman tace, ta hanyar abin da ya wuce kowanne gutsure hybridization da cloned DNA, niƙaƙƙun gaɓãɓuwa ko roba rediyoaktif yi bincike ne a kula, wanda zai zama daidai da kowane gwajin samfurin. Idan ka canza wuri, ko tsawon idan aka kwatanta da bincike, idan wani sabon guntu ko bace - duk wannan ya nuna cewa da bincikar gene ya halartar gyara a cikin nucleotide jerin. Akwai takwas na asali dabaru na kwayoyin kayyade nazarin: jerawa (tabbatar da dalilin da DNA jerin), polymerase sarkar dauki (karuwa a yawan jerin), da shiri na primers da aka sani da kwayoyin halittu, DNA cloning, samar da recombinant kwayoyin samu sunadaran saboda recombinant kwayoyin, samar da cikakken sa (tarin library) cloned da wani ɓaɓɓake da aka samu ta hanyar yin amfani da ƙuntatawa.