Molecular genetic method yekuongorora

Kuti udzidze uye uone kusiyana-siyana kwechimiro cheDNA, ma molecular genetic method inoshandiswa. Kune imwe nzvimbo yeDNA iyo inoongororwa, iyo nzvimbo inonzi chromosome, geni kana kuti iyo, nzira dzakasiyana. Pakati pemwoyo yese yemakemikari-genetic method inosanganisira kushandiswa kweRNA uye DNA. Zvose izvi nzira dzakaoma kwazvo, pasina ma laboratori mamiriro ezvinhu haagoni kuitwa, uye vashandi vanofanira kunge vakakodzera. Iri basa rinoitwa mumatanho akawanda.

Nhanho

Kutanga, samples yeRNA kana DNA inoda kuwanikwa. Pano, iyo molecular genetic method inogona kushandiswa kune zvinyorwa chero chipi zvacho: donho reropa, leukocytes, tsika ye fibroblasts, muchembera mutsvuku (kunyora), kunyange bhudzi remvere, - DNA inogona kuwanikwa kubva kune imwe yamuenzaniso. Izvo zvakakodzera kushandisa chero masero ega genetic method uye zvakasiyana-siyana zvakasiyana, uye kakatogoverwa DNA inochengeterwa kwenguva yakareba munguva yehuvhuta. Chikamu chechipiri chakazvipira kune kuunganidza zvidimbu zvakakosha (amplification) yeDNA, kuti ipe iyo inobatsira kutunga polymerase in vitro (in vitro, pasina kubatanidzwa kwehupenyu hupenyu). Somugumisiro, tsananguro yeDNA yakasarudzwa inowedzera nekugadzirwa kweketani iyi, uye chiyero cheDNA chinowedzera chaiyo mamiriyoni nguva.

Chikamu chechitatu chema molecular genetic nzira yekuongorora ndechokudziviswa kweDNA yakawanda (uku kupatsanurwa, kubvarura kana kucheka). Kurambidzwa kunoitwa uchishandisa polyacrylamide kana agarose gel electrophoresis. Iyi molecular genetic method yekudzidza DNA inobvumira chikamu chimwe nechimwe kuti chive nechinzvimbo mune gel. Mushure mezvo, gel rinorapwa ne ethidium bromide, iyo inokwanisa kusunga muDNA, ultraviolet irradiation inoitwa, mushure mezvo zvinogona kuitika munzvimbo dze luminescence. Maitiro ezvibhedheni zvekuongororwa dzakasiyana uye dzakawanda, zvisinei, matanho maviri okutanga anowanikwa kune vose. Asi kuti uone zvikamu zveDNA, gel rinogona kusvibiswa nemimwe nzira dzakawanda dziripo.

Mhando

Nzira dzakajeka uye dzakapararira dzekuona mycobacteria dzinosanganisira pamusoro apa-yakatsanangurwa ma molecular genetic method yeDNA tsvakurudzo. Chinokosha chaicho ndechekuona muzvinyorwa zvekutsvaga zvidimbu zvakasiyana zveketani yezvirwere zveDNA. Nzira dzezvemakemikari dzekuongorora dzinenge dzisina nzira inobatsira yekuziva chirwere chakadai sechirwere cheshuga. Uchishandisa polymerase chain reaction (PCR), unogona kuva nechokwadi chokuti DNA yepakutanga ichawedzera nhamba yemakopi mumamiriyoni, ndiko kuti, kuchava nekusimudzirwa, uye izvi zvichabvumira kuona mhinduro. Nzwi rekunzwisisa pano rakanyanya kwazvo-pamusoro pe makumi mapfumbamwe nemashanu kubva muzana, iyo ndiyo inonyanya kushandisa nzira iyi.

Zvose zvema molecular genetic nzira dzekutsvakurudza dzinowanzobudirira sezvinyorwa kudzokorora, sezvo munyaya ino shanduro yerimwe sarudzo inoratidza hutano chaiyo ye oligonucleotide yakawedzera nemazana nematanhatu. Kunyange tsika inosanganisa tuberculosis yomuviri wekupumha yakaderera zvikuru mukushushikana kwayo. Ndicho chikonzero mishonga yemazuva ano inobva pamatomu ekuberekwa kwema genetic ekuongorora hutachiona. Uye iyo yakatsanangurwa nzira inonyanya kushanda pakusangana nemakiriniki e-antigenic yakasiyana-siyana, izvo zvakaoma zvikuru kugadzirisa neimwe nzira - mharidzo yehutano yakakosha uye nguva yakareba yekurima inodiwa. Nyanzvi yemasikirwo uye maitiro emagenethi anopa maitiro akasiyana zvachose.

Kuongororwa kwechirwere chetachiona

Vanovaka zvirongwa zvePCR zvechirwere chetachiona kakawanda vachishandisa izvozvi DNA zvinogadziriswa izvo zvakananga kumarudzi ose ezvirwere izvi. Kuti uwane chinangwa ichi, zvinyorwa zvinotarisa kuwirirana kweZVA zvikamu (IS-986, IS-6110) zvinowanzoshandiswa, sezvo izvi zvinongoramba zvichifamba zvinongoratidzira mhando dze mycobacteria ye tuberculosis uye nguva dzose dziripo mumakopi akawanda mumagenome. Uyezve, DNA inogona kusarudzwa kubva kune tsika dzakachena uye kliniki (sputum yevarwere) neimwe nzira inogamuchirwa. Somuenzaniso, pane nzira yeBoom, iyo inoshandisa lysis buffer inobva ku guanidine, silika uye thiocyanate semutakuri weDNA. Nhamba yevarwere vane chirwere chinokonzera bhakitiriya yakanyanya kuwedzera gore negore, saka saka yakasiyana zvakakwana sangano rakasimbisa pachayo mumagariro echechi: ma molecular genetic nzira yekudzidza DNA ine basa guru mukuongorora.

Zvisinei, tinofanira kubvuma kuti hazvisi zvisina kukanganisa zvimwe. Kushandiswa kwepurogiramu yePCR kunowanzounza huwandu hwakawanda hwezvakanaka zvenhema, uye mhosva apa haisi kungoita zvisiri izvo chete, asiwo humwe hutsika hweyo nzira pachayo. Pakati pezvimwe zvinhu, kushandisa nzira iyi yekuongorora, hazvigoneki kutarisa kuwanda kwehupenyu hwemycobacteria inowanikwa. Asi kukanganisa uku hakusiko kunokosha. Maitiro ezvemakemikari ekuzivikanwa kwePCR anosanganisira dambudziko rekusvibiswa kweDNA mycobacterial. Chiratidzo chinodiwa nekuda kwechikonzero che PCR ma laboratories chakanyanya kugadzikana, zvinoda kuvapo kwezvikamu zvitatu zvakatsaurwa. PCR teknolojia ndiyo yemazuva ano uye yakaoma kwazvo, kushandiswa kwayo kunoda midziyo yakakwana nevashandi vakadzidziswa zvikuru.

Bacterioscopy

Panosimbisa kuongororwa, zviitiko zvekudzidza kwePCR zvinofanira kunge zvichifananidzwa nezvose zvakasara: kliniki yekuongorora, radiography, smear microscopy, kudyara uye kunyange kupindura kune kurapa kwakakosha pano. Muchikamu chino chekuongorora, PCR inongova imwe yezvinhu. Tsvaga pathogen pakutanga kwekuongororwa kunogona kuva nzira dzakanakisisa uye dzinokurumidza - mabhakitiriya.

Pano tinoshandisa microscope chiedza (Tsiol-Nielsen ruvara) uye luminescent (fluorochrome coloration). Kubatsira kwebhakitiriya inokonzerwa nekukurumidza kunoguma. Ikusavhiringidza kwayo kunofungidzirwa kuva mikana isina kukwana nekuda kwekunzwika pasi. Zvisinei, iyi nzira inopiwa kuHIV kukurudzira seyo mari yakanyanya uye inokosha yekuziva varwere vane tuberculosis. Kuziva mycobacteria nemaitiro eBacteriological kune zvakakosha zvekufananidzira, uye kusunungurwa kwebhakitiriya kunoshandiswa. Maitiro ezvemakemikari ekudzidza chirwere chetachiona anonyanya kuvimba neizvi.

Tsvakurudzo yedzidziso

Kunyanya kuzivikanwa kwemycobacteria kunonzwisiswa nedzidzo zvedzidzo. Kudyara kwezvinyorwa zvinoshandiswa mumazai evha: Mordovsky, Finn II, Levenshtein-Jensen nezvimwe. Chiratidzo chekusimudzirwa kwekudzivirira kwe mycobacteria kune zvinodhaka uye humwe humwe uchapupu hwekubudirira ndehuwandu hweyangukobacteria kana mazana avo mu vitro kana nzira yekutsika inoshandiswa. Kuwedzera huwandu hwekugoverwa kwe mycobacteria, chirwere chechirwere chinodyarwa kune mimwe mitambo.

Kugutsa zvido zvetsika dzakawanda, iyo causative agent inowanikwawo nemhepo inoshandiswa. Panguva imwecheteyo, maitiro ezvinyorwa zvemabhuku ekuwedzera kwechirongwa cheVANTES anoshandiswa. Miti inofanira kuve yakabatanidzwa kusvikira masvondo manomwe kusvika masere. Nenguva ino, kudyara nekushaikwa kwekukura kunogona kutaridzirwa kusina kunaka. Nzira yakanakisisa yekuziva kuti mycobacteria tuberculosis isuongorudzo dzehupenyu: dzinobata zvirwere zvekugovera zvipembenene, izvo zvinonyanya kugadzikana nehutachiona.

Nhamba shomanana

Nzvimbo inofadza zvikuru yekutsvakurudza, iyo yakawanikwa kuburikidza nekuongororwa kwePCR, yakanga iri kudzidza kwaMer tuberculosis, chirwere chinopera. Iko pfungwa yezvino yehutachiona hwehutachiona inoratidza kuti kune vanhu zana vave vachibatana naM M. tuberculosis, makumi mapfumbamwe vanogona kutapukirwa, asi gumi chete avo vane zvirwere zvinoshanda. Izvo zvose zvine utachiona hunopesana, uye naizvozvo mune makumi mapfumbamwe kubva muzana zvezvirwere hutachiwana hucharamba huchiri kure. Yakanga iri iyo molecular genetic method yakabatsira kuona ichi.

Genetics inopesana kuti makumi mashanu neashanu kubva muzana yevaya vane tsika dzisina kunaka uye makumi masere kubva muzana yevaya vane utachiona hweM. tuberculosis, asi nechirwere chisiri chemavara, vakagamuchira mhinduro dzakanaka dzePCR. Yakanga iri maitiro ehutachiona akazobatsira kuziva varwere vari mumakakatani achishandisa masangano ePCR, uye migumisiro yekuongorora kwavo (microscopy nezvirimwa) zvakanga zvisina kunaka, uye hutachiwana hwehutachiona hwaMer tuberculosis hwaivepo.

Ongororo yemazuva ano

Bacteriological laboratories yeRussia Federation vanoshandisa nzira yakakurumidza yekufungisisa zvakakwana: nitrate reductase basa re mycobacteria rinoshandiswa nenzira yeGriss reagent. Anti-tuberculosis nzvimbo dzinoshandisa nzira inobvumira kutsanangura kusagadzikana kwezvinodhaka. Ichi chirimwa mumasikirwe emvura, apo radiometric uye fluorescent system yekutumira kukura kwemycobacteria inogadziriswa. Kuongorora kwakadaro kunoitwa nokukurumidza - kusvika kumavhiki maviri.

Parizvino, nzira itsva dziri kuumbwa: kushandiswa kwezvinodhaka zve mycobacteria kunoongororwa pajenotype level. Kuongorora maitiro emakemikari ekurwisana kunoratidza kuvapo kwemajini mu mycobacteria. Aya mazamu anobatanidzwa nekudzivisa mamwe mishonga. Somuenzaniso, mazamu kasA, inhA, katG zvinopesana ne isoniazid, rpoB ku rifampicin, 16Sp RNA uye rpsL ku streptomycin, kusvika ku ethambutol, gyrA kusvika fluoroquinolone, nezvimwe zvakadaro.

Kuchinja

Muchizivo chemazuva ano, ma molecular genetic level yeDNA nzira yakakura zvikuru uye yakabvumirwa kuita zvidzidzo zvakakura zvekuchinja-chinja munharaunda yavo yose. Iye zvino tava kuziva kuti kuchinja mutsva mu 516, 526 uye 531 codons yeRpoB gene yakawanda, uye kushorwa nemishonga yakasiyana-siyana kwave kuonekwa. Kune nzira dzakasiyana-siyana dzekunyora mycobacteria kushandisa kwete chete tsika dzechigarire - biochemical, biological uye netsika, asi mazuva ano masikirini emagene anowanzoshandiswa zvikuru. Iko kune iko kwakakwana uye nzira dzekutsvaga dzinovimbika dzekucherechedza zvirwere zvemaoko. Izvo zvinobva paDNA tsvakurudzo munharaunda chaiyo yeimwe geni. Izvi zvinowanzotora nzira yakaoma, nguva inopedza uye inodhura, asi iyo inopiwa nedzimwe nzira dzema molecular genetic analysis dzakanyatsojeka uye dzinodzidzisa kudarika dhenda yezvimwe zvose kuongororwa.

Iyo yakave ichizivikanwa kwenguva refu kuti DNA haishanduki pamusoro pehupenyu hwose hwemuviri, kuti iri mune imwe nuclear nyukliya, zvisinei, uye izvi zvinoita kuti zvikwanise kutora zvachose masero emuviri kuti aongororwe, pane zvikamu zvese zvepenyika. Rimwe jena rakakanganisika rinogona kuonekwa musati maonekwa yekutanga zviratidzo, pamberi pekliniki yakaonekwa yechirwere, uyewo mune vanhu vane hutano hwakanaka hwehutano, asi vane kuchinja mutendi. Maitiro ezvemakemikari ekuongororwa kwezvirwere zvinogona kuonekwa neDNA-diagnostic approach, uyewo nekuongorora kusarura kwechirwere mumhuri ine marker DNA loci (polymorphic sites) iyo yakabatana zvikuru nejeri rakakanganisika (kureva, nzira DNA isinganzwisisiki yekuongorora). Kutungamira kana kusananga - chero DNA inoongororwa inobva kune nzira dzinoratidza nzvimbo yakatsanangurwa yeDNA yevanhu.

Tarisa nzira

Nzira dzakananga dzeDNA dzinoongororwa dzinoshandiswa mumamiriro ezvinhu apo geni-chinokonzerwa nechirwere chinotaridzika chinonyatsozivikanwa, uye mhando dzekuchinja kwayo dzinozivikanwawo. Somuenzaniso, nzira dzakananga dzakakodzera kune zvirwere zvakasiyana-siyana. Iyi ndiyo Huntington's chorea (kukura kweKTG kudzokorora), phenylketonuria (R408W), cystic fibrosis (delF508, kuchinja kukuru) nezvimwe. Nzira huru yekushandisa nzira yakananga ndeye zana zana kunyatsoziva kwekuongororwa, uye hapana chikonzero chekuongorora DNA yevamwe vemhuri. Kana kushanduka kwemajini kunowanikwa, kunokubvumira kunyatsosimbisa kuonekwa kwehutano, sarudzai genotype yevamwe vemhuri yakamanikidzwa.

Rimwe ruzivo rwekutsanangurira zvakananga ndiko kuonekwa kweheterozygous carriage yekuchinja kwakashata mumhuri uye vabereki vemunhu akafa kubva pachirwere. Izvi zvinonyanya kuitika kune zvirwere zvekutsvaga kwezvokudya. Kukanganiswa kwezvirevo zvakananga zviripo zvakare. Kuti uite mazviri, iwe unofanirwa kuziva zvakajeka kugona kwechirwere chechirwere, iyo exon-intron yakarongeka uye inoratidzirwa yekuchinja kwayo. Hasi dzose zvirwere zvemonogenic dzakagamuchira mashoko akadaro nhasi. Kuziva kushandiswa kwenzira dzakananga hakugoni kuonekwa sekuzara, sezvo geni rimwechete rinogona kuva nehuwandu hwekuchinja kwehutano, iyo inofanirwa kukura kwezvirwere zvechizvarwa.

Nzira dzisinganzwisisiki

Nzira dzisinganzwisisiki muDNA dzinoongororwa dzinoshandiswa mune dzimwe mamiriro: kana iyo yakaora geni isingazivikanwi, asi inongogariswa panzvimbo yechromosome, kana kana kuongorora zvakananga hakuna kubereka mitsara (izvi zvinoitika kana geni ine sangano rakaoma maserosi kana urefu hwakareba, kana yakawanda mairi Kuchinja kwechipatara). Nzira dzisinganzwisisiki dzinoshandiswa kuongorora kusarura kwepolymorphic markers mumhuri ye alleles. Markers vari munzvimbo imwe chete yechromosomal kana kuti vanobatana zvikuru neyemusha wechirwere uye vanomiririra kubvisa kana kuiswa, kumwe kunyorera, kudzokorora, uye kuporesa kwavo kunokonzerwa nenhamba dzakasiyana mubato rezvinhu.

Izvo zvakanakisisa zvekusaziva zvakananga microsatellite uye minisatellite polymorphic markers, iyo inowanzoparadzirwa mujeni revanhu. Kukosha kwavo kunoratidzwa mukunyatsoziva, kana mararamiro emagetsi pakati pekukuvadza mujeni uye mucherechedzo hausi mukuru. Muchikamu chekupedzisira, kunyatsoratidzwa kwekuongororwa kunotsanangurwa kusvika pamwero wakawanda nehuwandu hwekugadziriswa pakati pe polymorphic marker uye kukanganisa. Nzira dzisinganzwisisiki dzekuongororwa dzinopawo chinangwa chekutanga chekudzidza kwemazuva ose evanhu vakaongororwa pakati pevatakuri vekuchinja uye varwere, pamwe nekudiwa kwekuona mikana yekurwara nehutachiona uye kugadziriswa kwekubatanidza kwemavara uye zvinyorwa zvese zvinoshandiswa zvejeni.

Dzimwe nzira

Zvishoma zvikamu zveRNA kana DNA, pamwe chete nejeni rimwe chete hazvikwanisi kuonekwa nezvinyorwa zvinyoronyoro, naizvozvo, kuitira kuti tizive kuchinja-shanduka, nzira dze molecular genetic diagnostics dzinodiwa. Izvo zviripo "Human Genome Project", sezvimwe zvakabudirira mu molecular genetics, zvine nzira dzakawanda dzakapararira mukana wekuona zvirwere zvinokonzerwa nehutano - zvose zvisati zvaitika uye shure kwekuberekwa. Idzi nzira dzinogona kupa nekukurumidza kuona nekuita kufanotaura kwezvirwere zvakasiyana-siyana uye zvinogadziriswa, izvo zvinotanga kuitika pakukura. Zvinosuruvarisa, maererano nehutano hunyanzvi, maitiro emagenheti zvidzidzo dzimwe nguva anopfuurira mberi kwehutsika hunosimbiswa maererano nekuremekedza, kunyanya kana kuongororwa kunoitwa mukuyaruka uye muhuduku.

Structural uye kusanganiswa chromosomal abnormalities ndivo vakawanda zvinokonzera chirwere uye kenza, uye malformations zhinji. Chromosomal aberrations vanofanira kuzvibatanidza, izvo zvinokosha mhuri mazano - rokuongorora for, pamwe dzokubereka yokubatwa pamuviri ramangwana. Mashoko okutodza kuongorora ndiyo "nzira yemandorokwati" yokugara nechirwere, asi zviri zvishoma. Chete nzira samolecular nemagene pachisara kuita zvakawanda, nokuti akashandisa cloning zvemichina yakavakirwa fluorescent Labels anokwanisa yavo yakakwirira nehanya kuziva dzisinganyatsooneki chromosomal kuchinja kuti hazviitiki kuona dzenhoroondo cytogenetic zvidzidzo. unyanzvi izvi vari kuramba kuwedzera kwedu chirwere nezvaanogona, kana akaongorora, vana vakaremara zvokukura, mupfungwa retardation, zvimwe zvirwere zvakawanda nhaka.

zvakawanikwa

Zvakakosha chaizvo kuti vanhu vakanga majini marongerwo uye kushanda ruzivo, mhando variability, mano okuona nhaka zvirwere kwakaitika panyaya kukura samolecular genetics. nzira yayo chinangwa pakudzidza morekuru reDNA - uye kana kuipa, kana wakaremara. Kugadzirira deoxyribonucleic asidhi sequences pamusoro nucleotides (DNA) kunosvika mumatanho nokugamuchira ivhu kuziva mumwe zvimedu. Voga pakati genomic DNA mumasero, yairambidzwa (kubvambura), amplification (cloning), electrophoresis nezvimedu (kupatsanura mutariri wavo yemagetsi uye samolecular uremu kubudikidza agarose gel). Kuziva chaizvo zvimedu iri pamusoro pechiso munhu discrete mutsetse.

Zvadaro achipinda chiito chaiwo filters, kuburikidza icho chinopinda mumwe kukamuka hybridization pamwe yakaumbwa DNA nezvimedu kana chakaita zvokugadzirwa radioactive Yatanga Kuongorora iri kudzorwa, izvo zvichava akaenzana mumwe bvunzo mharidzo. Kana kushandura chinzvimbo kana urefu zvichienzaniswa vachiongorora, kana chaenga mutsva kana akanyangarika - ichi zvose kunoratidza kuti anoongororwa Gene kwave kuchiita kugadzirisa ari nucleotide kutevedzana. Pane sere mitoo inokosha samolecular nemagene zveBhaibheri: sequencing (Kutsunga DNA kutevedzana), polymerase cheni maitiro (nokuwedzera kwenhamba sequences), kugadzirira primers anozivikanwa majini, DNA cloning, kugadzirwa recombinant mamorekuru hwokurara mapuroteni nokuda recombinant mamorekuru, ndinosika yakakwana yakatarwa (muunganidzwa raibhurari) yakaumbwa zvimedu avapo achishandisa chengeto.