Yamamolekhula zofuzo ucwaningo indlela

Ukuze uhlole futhi ukukhomba ezahlukeneyo e isakhiwo DNA esetshenziswa yamangqamuzana indlela zofuzo. Ukuze esifundeni DNA ngalinye, okuyinto ihlola kule ndawo ye-chromosome, isakhi sofuzo noma allele, izindlela kuhluke. Ezicashile indlela ngayinye yamangqamuzana zofuzo yakhiwa abanye noma ezinye zokukhwabanisa RNA ne-DNA. Zonke lezi zindlela zinezikhathi i liyinkimbinkimbi kangakanani, ngaphandle kwezimo zelabhorathri ayikwazi kwenziwe, kanye nabasebenzi kumele kube abaqeqeshiwe. Lo msebenzi wenziwa izigaba eziningana.

izigaba

Okokuqala, amasampula RNA noma DNA lekufanele akhicitwe. Lapha, indlela yamangqamuzana zofuzo singasebenza cishe noma yikuphi okubalulekile: a iconsi legazi leli, leukocyte, fibroblasts isiko, mucosa (esusa), ngisho follicle izinwele, - DNA ingatholakala kusuka kunoma iyiphi isampula. Kuyinto efanelekile ukusebenzisa noma isiphi izindlela zofuzo ezinto ongakhetha zabo ezihlukahlukene futhi baye DNA eside isehlane lilondolozwe eqandisiwe. Isigaba sesibili lunikezelwe ekuqongeleleni izingcezwana oyifunayo (okhulisa) kwe-DNA, njengoba kusiza ukuqinisekisa ukuphendula polymerase chain ngaphandle kwesibeletho (ngaphandle kwesibeletho ngaphandle ukubandakanywa ophilayo umzimba). Ngenxa yalokho, ezikhethiwe DNA isinqamu liphindaphindeke yilo ukusabela chain, kanye nenani DNA inyuka safinyelela izigidi isibili izikhathi.

Isinyathelo sesithathu ku izindlela ukucwaninga ngofuzo yamangqamuzana Kusolwa andisa izivimbelo DNA (lokhu kwehlukana, edwengula noma ahlabayo). Okuvumelekile eyenziwa electrophoresis kwi polyacrylamide noma ijeli agarose. Le ndlela yamangqamuzana-genetic ukutadisha isinqamu DNA ivumela wonke umuntu ukuthatha likuphi ijeli. Ngemva kwalokho, ijeli ziphathwe ethidium bromide, uyakwazi sesibopho ukuthobisa DNA, ngemisebe nge ultraviolet, ke kungenzeka ukuba bagcine luminescence izingxenye. izindlela Yamamolekhula zofuzo zokuxilonga zihlukene futhi eziningi, kodwa izinyathelo ezimbili zokuqala evame ukubuzwa yibo bonke. Kodwa ukuze abone izingcezwana DNA, ijeli kungenziwa ezinemibala, futhi eziningi nezinye izindlela ezikhona.

zinhlobo

Izindlela ngqo kakhulu futhi kusakazeke uzingela mycobacteria kungafaka ngenhla yamangqamuzana DNA yezakhi zofuzo yokufunda indlela. essence yayo wukuthi, ukuze abone scan uchungechunge impahla imvuthuluka ezithile DNA kwamagciwane. Yamamolekhula zokupopola zofuzo Awunayo okwamanje indlela eyanele ukuba aqaphele izifo ezifana nesifo sofuba. Ukusebenzisa ukusabela polymerase chain (i-PCR), ungaqiniseka ukuthi DNA yasekuqaleni kuzokhuphula inani lamakhophi e izikhathi eziyisigidi, okungukuthi, lapho kuyoba okhulisa, futhi ngeke sibonise imiphumela. Izinga ukuzwela kakhulu - ezingaphezu kuka asithoba anamihlanu amaphesenti, okuyinto inzuzo enkulu yale ndlela.

Zonke ezinye izindlela yamangqamuzana-genetic ucwaningo mayelana nokusebenza kahle isivuno amakhophi amaningi ngokoqobo kabili, kusukela kulokhu isampula kwakha luhlaka sikhombisa oligonucleotide thize anda komunye ezingamakhulu ayisithupha izikhathi. Ngisho uphethwe isiko sofuba umgudu wokuphefumula kakhulu yehlisa ukuzwela yayo. Yingakho imithi yesimanje kusekelwe izindlela yamangqamuzana zofuzo ukuxilongwa kwesifo sofuba. Indlela okuchazwe ephumelela kakhulu lapho sisebenzelana Amagciwane ephezulu kokuhlukana antigenic, ukunquma ukuthi enye indlela kunzima kakhulu kangakanani - kudinga ngokukhethekile abezindaba izakhi futhi uhlakulele isikhathi eside. izindlela kwamakhemikhali kanye yamangqamuzana zofuzo ukukhiqiza umphumela ezahlukene kakhulu imiphumela.

uphethwe isifo sofuba

Marshall PCR uphethwe isifo sofuba ngokuvamile usebenzisa labo DNA ukuthi acace zonke izinhlobo ezine lesi sifo. Ukuze ufinyelele kulesi ngokuvamile zisebenzisa kwenziwa ukuthi ukubona ukulandelana IS izakhi (IS-986, IS-6110), njengoba lezi zinto elichaza zinhlobo ezifudukayo kakhulu sofuba Mycobacterium futhi njalo samanje amakhophi amaningi ufuzo. Futhi-DNA isizinda kungaba kwenziwe letiphatselene nemasiko okumsulwa kanye Clinical (isikhwehlela iziguli) nganoma iyiphi enye indlela efanelekayo. Ngokwesibonelo, lapho Boom indlela lapho sesikhashana lysis isetshenziswa esekelwe thiocyanate guanidine futhi Silica njengoba DNA yenethiwekhi. Inani leziguli ukuthi kuhluke bacteriological abampofu okwandisa minyaka yonke, futhi ngenxa yalokho umkhuba emtholampilo isungule ezingeni ohluke ngokuphelele inhlangano: indlela yamangqamuzana-genetic ukutadisha DNA iye lunendima eyinhloko e uphethwe.

Nokho, kumelwe ngivume ukuthi akusiyo ngaphandle nemiphumela ethile engathandeki. I-PCR ndlela ukusetshenziswa ngokuvamile kuletha lenqwaba Imiphumela zamanga-HIV, futhi isizathu akuyona amaphutha lobuchwepheshe kuphela, kodwa futhi izici indlela uqobo. Ngaphezu kwalokho, besebenzisa le ndlela ukuxilongwa ukucacisa ukusebenza mycobacteria, okuyinto sezihlonziwe, kumane nje akunakwenzeka. Kodwa lokhu kusimo akulona ezibaluleke kakhulu. izindlela Yamamolekhula kwezakhi PCR ukuhlola kuhilelani ingozi ukungcoliswa DNA mycobacterial. izidingo Certification yalesisizathu for laboratories PCR yenzelwe kuphela kanzima, zidinga izakhiwo ezintathu ezihlukene. PCR ubuchwepheshe kuyinto yesimanje kanye eziyinkimbinkimbi kakhulu, kudinga ukusetshenziswa imishini ezifanele kanye nabasebenzi ephezulu oqeqeshiwe.

bacterioscopy

Lapho imiphumela sitholakele ukuhlaziywa kumele, uma iqhathaniswa nezinye idatha: luhlolo yomtholampilo, esimweni, smear microscopy, isivuno ngisho ephendula umuthi othile abaluleke kakhulu. Kulesi chungechunge, PCR izifundo ingenye yezingxenye kuphela. Thola pathogen ku ekuhlonzweni kungaba izindlela elula futhi esheshayo - bacteriological.

Kukhona isetshenziswa ngesibonakhulu ukukhanya (umbala Ziehl-Neelsen) kanye Fluorescent (Umbala fluorochromes). Inzuzo kuyinto ngesivinini smear imiphumela. Kodwa ethile yayo ngokufanelekile njengezidalwa ezingaphezu kwabantu amandla elinganiselwe ngenxa ukuzwela ongaphakeme. Nokho, le ndlela kunikezwa UBANI izincomo njengoba ukonga kakhulu futhi phansi ukuthola iziguli zesifo sofuba. Ukuthola mycobacteria indlela bacteriological has ukuqagela ukubaluleka ecatshangelwayo excretion quantitatively virus. unethemba Okuningi ukubhekana naso yamangqamuzana izindlela zofuzo ucwaningo isifo sofuba.

Kwamasiko

ukuthola kangcono mycobacteria ukuqaphela izifundo zamasiko. Ukuhlwanyela impahla sokugembula esenziwe phakathi naphakathi Iqanda: Mordovsky, uFinn II, LJ, nokunye okunjalo. Zokubekisa ukumelana mycobacteria emithini futhi ubufakazi ongaqondile ukusebenza eziningana mycobacteria kanye amakoloni zabo ngaphandle kwesibeletho, uma indlela laloko isiko ucwaningo. Ukuze sokukhulisa iphesenti kwemihlambi mycobacteria inoculation of impahla sokugembula ligcinwe ezindaweni eziningi.

Ukwanelisa izidingo eziningi kwamasiko, lwegciwane kuhlanganise isibonelelo nasoketshezini. Ekwakhiweni kwalesi simiso futhi ezenzakalelayo metering uhlobo VANTES ukukhula. Izitshalo kumele ibanjelwe bayashintshana ekufukameleni kufika eziyisikhombisa kwabayisishiyagalombili amasonto. Ngalesi sikhathi lesi sitshalo nokuntula ukukhula angabhekwa ezimbi. Indlela ephumelela kunazo ukuthola sofuba Mycobacterium cabanga amasampula yemvelo: zokuxilonga ngegciwane impahla Guinea izingulube, okuyinto singangena kakhulu TB.

Ezinye izibalo

ensimini abathandwayo zokutadisha, nakho wavulwa PCR ukuhlola kwaba ukutadisha M. sofuba - ukutheleleka esicashile. Umqondo yesimanje ye-TB kusikisela ukuthi aphume abantu abayikhulu ababe nabo M. sofuba, ayisishiyagalolunye kungenzeka itheleleke, kodwa kuphela eziyishumi kulo bayizinkulungwane isifo asebenzayo isakhiwa. Abanye babe amasosha omzimba-TB, futhi ngoba amaphesenti ayisishiyagalolunye amacala ukutheleleka uhlala esicashile. Thola iphethini kubasizile indlela yamangqamuzana zofuzo.

Izazi zezakhi zofuzo bathi amahlanu nanhlanu angu labo impahla izitshalo zabo sokugembula kwatholakala ukuthi azinaso, futhi abantu abangamaphesenti angu-utheleleke nge M. sofuba ayisishiyagalombili, kodwa eligeleza kungekho ukubonakaliswa radiographic zezifo, PCR izimpendulo omuhle wathola. Kuyinto indlela zofuzo zokuxilonga wasiza ukukhomba iziguli engozini ngu-PCR izifundo, kanye imiphumela cwaningo yabo (microscopy namasiko) abangu ongathandeki, futhi ukutheleleka subclinical M. sofuba wayekhona.

nocwaningo lwanamuhla

I Russian Federation futhi laboratories bacteriological zisebenzisa indlela esasizosheshisa ka lokuhlala ngokuphelele: nitrate reductase umsebenzi mycobacteria ihlolwe ngu Griess reagent. izikhungo elwa ne-TB zisebenzisa indlela evumela ukucacisa yokumelana nomuthi. Lokhu zokutshala media liquid, lapho okuzenzakalelayo radiometric futhi Fluorescent accounting system ukukhula mycobacteria. azihlole kwenziwa ngokushesha - amasonto amabili.

Okwamanje, izindlela ezintsha isakhiwa: izidakamizwa ukumelana mycobacteria kukalwa ezingeni genotype. Isifundo etindlela yamangqamuzana ukumelana izakhi zofuzo kanye ikhombisa khona e mycobacteria. Lezi izakhi zofuzo elihlobene ne amandla okumelana nemithi ethile. Ngokwesibonelo, Kasa ufuzo, inhA, katG amelana isoniazid, rpoB isakhi sofuzo - rifampicin 16Sp RNA izakhi zofuzo kanye rpsL - i-streptomycin, emb1 - ukuze ethambutol, Gyra - a fluoroquinolone nokunye.

ukuguqulwa kwezakhi zofuzo

Imiphumela yaloku kuhlola okungenhla yesimanje khudlwana yamangqamuzana-zofuzo ezingeni indlela ukutadisha DNA futhi bavunyelwe ukuba afeze izifundo ezinkulu yokwanda koshintsho zonke bohlu lwabo. Manje siyazi ukuthi ukuguquka kwezakhi zofuzo ezivame kakhulu kuleli 516, 526 kanye 531 codons isakhi sofuzo rpoB, futhi bahlonze lezi amandla okumelana nemithi ehlukahlukene. Kukhona lahlukene izindlela ukuze ukuthayipha ka mycobacteria usebenzisa izindlela zomdabu hhayi kuphela - kwamakhemikhali, begazi namasiko, kodwa futhi kusetshenziswa kakhulu amasu zofuzo yesimanje yamangqamuzana ezinto eziphilayo. Kakade kukhona eyanele futhi ahlinzeke lengiyo uphethwe indlela ukuthola izifo monogenic. Zisekelwe izifundo DNA endaweni esiqondile isakhi esithile. Lokhu kuvamise Lena inqubo eyinkimbinkimbi, isikhathi esiningi futhi kuyabiza, kodwa idatha olunikezwa ukuhlaziywa yamangqamuzana zofuzo, kodwa linembile lapho likhuluma kakhulu futhi ulwazi kuka idatha zonke ezinye cwaningo.

Sekuyisikhathi eside kwaziwa ukuthi uHlelo DNA ayishintshi lonke ukuphila umzimba ukuthi kunoma iyiphi nucleated amaseli odnakova, futhi lokhu kwenza kube nokwenzeka ukuba athathe ukuhlaziya ngokuphelele zonke emangqamuzaneni omzimba, kunoma yisiphi isigaba ontogeny. Isakhi ezilimele kungatholakala ngaphambi ukubonakala izimpawu zokuqala kuya full-scale isifo sokucindezeleka, kanye nabantu enempilo heterozygous, kodwa kokuba ngokuguqula izakhi zofuzo isakhi sofuzo. zofuzo lezinceku isifo izindlela Yamamolekhula zokuxilonga ukuvumela ukuba embule yayo (indlela eqondile, DNA-ukuhlola), kanye ukuhlaziya nokucwaswa lesi sifo emkhayeni nge marker loci DNA (polymorphisms zofuzo), okuyinto kuhlobene esakhini sofuzo ezilimele (ie, indlela engaqondile DNA-ukuhlola). Ngokuqondile noma ngenye indlela - yimuphi ukuhlola DNA kusekelwe izindlela yekubona ingxenye ngokuqinile kuchazwe we-DNA yomuntu.

izindlela ngqo

izindlela ngqo uphethwe DNA uma isoni isakhi sofuzo esiwufuzo is, eyaziwa ngokuthi eyaziwa kahle, futhi izinhlobo ukuguquka kwezakhi zofuzo zayo. Ngokwesibonelo, izindlela ezifanele oqondile eziningi izifo. Lokhu chorea Huntington sika (isandiso CTG-eziphindaphindiwe), phenylketonuria (R408W), cystic fibrosis (delF508, ukuguqulwa kwezakhi zofuzo eziyinhloko) nokunye okunjalo. Inzuzo enkulu indlela eqondile kuyinto ngokuphelele enganyelwe ukunemba zokuxilonga, futhi asikho isidingo ukwenza ukuhlaziywa DNA amanye amalungu omkhaya. Uma ngokuguqula izakhi zofuzo isakhi sofuzo elihambisana kutholakala, it evumela ncamashi agunyaze sitholakele ufuzo, genotype ukuzimisela ukuze amanye amalungu omkhaya sisindwa.

Enye inzuzo yokuhamba kwesikebhe uphethwe oqondile kubhekwa ukukhomba yenethiwekhi heterozygous yokwanda koshintsho kubi kusukela izihlobo nabazali owafa ngenxa yalesi sifo. Lokhu kuyiqiniso ikakhulukazi izifo autosomal esikhubazayo. Okumbi izindlela ngqo futhi ayatholakala. Ukuze sizisebenzise, kudingeka wazi kahle okwasekhaya isakhi sofuzo normal, exon-intron isakhiwo spectrum yayo futhi ukuguquka kwezakhi zofuzo zayo. Akubona bonke izifo monogenic namuhla Uthole ukwaziswa okunjalo. izindlela Informativeness oqondile ayikwazi kubhekwe ephelele, ngoba omunye futhi isakhi sofuzo esifanayo angase abe inqwaba kuguquka sokugembula esibangela ukuthuthukiswa izifo sitholwa njengefa.

izindlela engaqondile

izindlela ongaqondile e ukuhlola DNA bewasebenzisa, kwezinye izimo, uma isakhi sofuzo ezilimele is abaziwa, kodwa chromosomally kuphela, noma uma uphethwe umugqa enganikanga yi (kwenzeka, uma isakhi sofuzo eziyinkimbinkimbi inhlangano yamangqamuzana noma ngokwezinga elikhulu, uma kunezinhlobo eziningi ukuguquka kwezakhi zofuzo sokugembula). izindlela ongaqondile wenziwa ukuhlukaniswa ukuhlaziywa izimpawu polymorphic e allelic umndeni. Abamakayo ezitholakala okufanayo chromosomal esifundeni noma Locus kuhlobene eduze lesi sifo futhi bamele okususiwe noma okufakiwe, iphuzu izisusi, uphinda, kanye polymorphism yabo kungenxa inani elihlukile lamaseli block.

Iningi esilungele engaqondile uphethwe kubhekwe microsatellite futhi minisatellite polymorphisms, okuyinto zisakazwa zofuzo lomuntu. value abo obonakaliswe okusezingeni eliphezulu okuqukethwe ulwazi, uma ukulimala ibanga zofuzo phakathi umaka isakhi sofuzo ingenkulu kakhulu. Kulo mBhalo, ukunemba ukulinganisa kunqunywa ngokwezinga elikhulu ukuvama recombination phakathi umaka polymorphic futhi umonakalo. izindlela ongaqondile zokuxilonga futhi sinikeze saziso isinyathelo owandulelayo allele amaza we ihlaziye cwaningo labantu iziguli kanye abathwali yezinguquko, plus isidingo sokunquma ematfuba recombination ka nonequilibrium futhi adhesion nakomaka futhi alleles asabekayo.

ezinye izindlela

izingxenye iDemo RNA noma DNA, kanye isakhi sofuzo esisodwa ibukeke ngaphansi cwaningo ezincane akukwazi, Ngakho-ke, ukuze abone ukuguquka kwezakhi zofuzo adingwa uphethwe yamangqamuzana zofuzo. Kukhona "Human Genome Project ngokuthi", kanye nezinye kwentuthuko yezakhi zofuzo ezinwetshiwe kungenzeka izifo lezinceku - kokubili ngaphambi kanye nangemuva kokuzalwa komntwana. Lezi zindlela anganikeza kusenesikhathi futhi wenze poly- ukubikezela kanye izifo monogenic, ogama aseqala senzeka lapho usukhulile. Ngeshwa, ngenxa amakhono obuchwepheshe izifundo zofuzo yamangqamuzana ngezinye izikhathi ngalé kwemingcele yokuziphatha asethwe maqondana ifa, ikakhulukazi uma uphethwe entsheni futhi ebuntwaneni.

Takhi zezinombolo chromosomal kwezinso izimbangela ezivamile yesifo nayo umdlavuza, futhi kombungu eziningi. aberrations Chromosomal kudingeka abhekwe, okuyinto ebaluleke for ukwelulekwa umndeni - ukuhlola umhlahlo, kanye ingozi nekutala ukukhulelwa esizayo. Chromosome ukuhlaziywa iyona "izinga legolide" ukuxilongwa zofuzo, kodwa alinganiselwe. izindlela kuphela ukuhlaziywa yamangqamuzana zofuzo zingenza okwengeziwe, ngoba kukhona esetshenziswa cloning ubuchwepheshe esekelwe amalebula Fluorescent uyakwazi ukuzwela kwabo okusezingeni eliphezulu ukuhlonza chromosomal izinguquko ezinganakekile azinakwenzeka ukuthola classical izifundo cytogenetic. Lezi zindlela ngokwethukela ekwandiseni amakhono ethu zokuxilonga, lapho ehlolwa, nezingane ezikhubazekile zentuthuko, retardation ngokwengqondo, kanye nezinye izifo eziningi zenani elikhulu eziyifa.

okutholakele

Kubalulekile kakhulu esintwini kwakungamadodana kwezakhi zofuzo futhi umsebenzi ulwazi, izinhlobo kokuhlukana, ikhono lokuzwa izifo eyifa esasifakwa ngayo ukuthuthukiswa yezakhi zofuzo. izindlela zayo zihlose ekutadisheni molecule ye-DNA - nalapho kungokwemvelo, futhi uma kulimele. Ulungiselela deoxyribonucleic acid ukulandelana kwezinhlamvu (DNA) nucleotide ubabonisa ngezigaba kusukela ekutholeni amasampula ukuhlonza izingcezwana ngabanye. Kwemihlambi DNA ocwaningweni kusukela amaseli, Umkhawulo (Ukubhidliza), okhulisa (cloning), electrophoresis we zezingxenye ezithile (uhlukanisa ababaphathisiwe kagesi nesisindo yamangqamuzana by gel agarose). Ukukhomba izingcezwana ethize esisogwini ebusweni umugqa leyehlukene.

Khona-ke eba isenzo izihlungi ekhethekile, ngokusebenzisa oludlula isinqamu hybridization ngasinye bakha izingcezwana DNA noma zokwenziwa iphenya enemisebe kuyinto isilawuli, okuzokwenza ukuba alingane test ngayinye. Uma ushintsha isikhundla noma ubude kuqhathaniswa ne-probe, uma isinqamu entsha noma banyamalala - konke lokhu kubonisa ukuthi isakhi sofuzo ihlaziye usebhekene kabusha ngekulandzelana nucleotide. Kukhona eyisishiyagalombili amasu eziyisisekelo izifundo zofuzo yamangqamuzana: • Ukutshengisa ukulangazelela nokuthatheka (ukuzimisela DNA ukulandelana), polymerase chain reaction (ukwanda kwenani ukulandelanisa), ukulungiswa kwenziwa kwaziwa ufuzo, DNA cloning, amaphrotheni kwamangqamuzana emvelo isakhi sokuzakhela etholakala ngenxa molecule isakhi sokuzakhela, udala isethi ephelele (iqoqo umtapo) bakha izingcezwana zazitholakala ngokusebenzisa izivimbelo.