Mekhoa ea limolek'hule tsa likokoana-hloko ho etsa lipatlisiso le ho sebelisa ha bona

Limolek'hule tsa likokoana-hloko mekgwa ya ho etsa lipatlisiso phetha karolo ea bohlokoa ka moriana morao-rao, saense forensiki, le baeloji. Thanks ho tsoelo-pele ea ho ithuta ea DNA le RNA, motho eo o khona ho hlahloba liphatsa tsa lefutso tsa phelang beha moemeli causative, ho lemoha a lakatsa acid e nucleic ka motsoako oa acid, joalo-joalo

Limolek'hule-likokoana-hloko mekgwa ya. ha e le eng?

Morao ka 70-80s bo-rasaense ba e ne e le ba pele ba ho hlalosa se boleloang ke ho liphatsa tsa lefutso tsa botho. ketsahalo ena o ile a fana peleka ho ntshetsopele ya fetola liphatsa tsa lefutso le baeloji ea limolek'hule. Thuto ea thepa ea DNA le RNA e ne e bolela hore hona joale ho ka etsahala ho sebelisa acid tsena nucleic ka morero oa ho lefu tepelletse maikutlong, ho ithuta phatsa ea lefutso.

Ba lokisetsa ho ea DNA le RNA

Limolek'hule tsa likokoana-hloko mekgwa tekotshupo hloka qalang tse bonahalang: hangata e ena acid e nucleic. Ho na le litsela tse 'maloa ho khetha lintho tse bana ba tsoang har'a lisele tsa lintho tse phelang. E mong le e na le melemo ea eona le go tlhoka mesola, 'me e lokela ho nkoa e le ha a khetha mokhoa oa ho itšehla acid nucleic foromo bo hloekileng.

1. Ho lokisetsa tsa DNA ke Marmur. mokhoa li entsoe ka ho phekola motsoako joala dintho, precipitates sephetho le DNA bo hloekileng. The bothata ba ho mokhoa ona ke ho sebelisoa ha lintho tse senyang, phenol le chloroform.

2. itšehla thajana ea DNA ka boom boom ena. Ntho sehlooho tseo e sebediswa mona - ke guanidine thiocyanate (GuSCN). E khothalletsa batho ho deposition ea acid e deoxyribonucleic ka substrates khetheha, ho tloha eo e ka o ile bokella ka ho ba le pafara khethehileng. Leha ho le joalo GuSCN - ke sesitisa tsa TCP, 'me esita le karolo e itseng e nyenyane ea hore gets bukkake beha ka DNA e ka susumetsa tsela ea polymerase ketane itšoara joang, e leng bohlokoa ha ho sebetsa le acid nucleic.

3. pula ea litšila. mokhoa fapane le batho ba mehleng eo limolek'hule li ha ba beha acid e dehoksiribonukleinovoy le litšila. Ho etsa sena, sebelisa Ion le phapanyetsano. The bozangi litomba ke hore ha dintho tsohle ka rarolla.

4. 'Misa screening. Mokhoa ona o sebelisoa maemong a ha o ha ho hlokahale hore ba le boitsebiso bo nepahetseng mabapi le sebōpeho sa DNA molek'hule, 'me ho hlokahala hore a fumana ba bang ba dipalopalo. Lebaka le leng ke hore nucleic sebopeho acid e ka ho senyeha ha tšoarang detergents, haholo-holo alkalies.

Classification mekhoa ho etsa lipatlisiso

All mekhoa ea limolek'hule-likokoana-hloko tsa ho etsa lipatlisiso ba arotsoe ka lihlopha tse tharo tse ka sehloohong:

1. kholiso (sebelisa plurality tsa enzyme). Sena se akarelletsa ho PCR - polymerase ketane itšoara joang, e leng se phetha karolo ea bohlokoa ka ho ba bangata ba mekhoa e tekotshupo.

2. Neamplifikatsionnye. Sehlopha sena sa mekhoa e amanang ka ho toba le mosebetsi oa metsoako nucleic acid e. Mehlala ke 3 mefuta blots, ka situ hybridization, joalo-joalo

3. Mekhoa e thehiloeng ho amohelwa ha pontšo tswa fatang molek'hule, e tlama ho itseng DNA kapa RNA fatang. Mohlala - hybridization oona tsamaisong Hybride hapa System tharollo (hc2).

Enzyme e ka sebelisoa ka mekhoa ea limolek'hule tsa likokoana-hloko ho etsa lipatlisiso

Tse ngata dithekeniki limolek'hule tekotshupo akarelletsa tšebeliso ea mefuta e fapaneng sephara ea enzyme. Ka tlase ho sebelisoa hangata ka ho fetisisa:

1. thibelo potlakisang tsebetso ea lik'hemik'hale - "khaola" DNA molek'hule ho likarolo tse hlokahalang.

2. DNA polymerase - synthesizes e habeli-tsielehile deoxyribonucleic acid e molek'hule.

3. etsolla transcriptase (etsolla transcriptase) - sebelisoa ho synthesize DNA ho tloha e thempleite RNA.

4. DNA ligase - e ikarabella bakeng sa sebopeho sa litlamo phosphodiester pakeng nucleotides.

5. exonuclease - tlosa nucleotides tloha likarolo qetellong ea deoxyribonucleic acid e molek'hule.

PCR - mokhoa tsa motheo tsa DNA kholiso

Polymerase ketane itšoara joang (PCR) e ho pharaletseng sebelisoa ka baeloji ea kajeno ea limolek'hule. Mokhoa ona, oo ho oona e le 'ngoe DNA molek'hule ka fumanoa palo e khōlō ea likopi (ho hodisa molek'hule).

Mesebetsi e ka sehloohong ea PCR:

- phumano ya mafu a;

- cloning DNA likarolo, Gene.

Hore le phethe polymerase ketane itsoara joang ka hloka thepa ea motheo e latelang: ka lekhetlo la pele DNA molek'hule, e thermostable DNA polymerase (Taq kapa Pfu), deoxyribonucleotide phosphates (mehloli ea makalana naetrojene), le primers (2 selelekela 1 DNA molek'hule) le ka boeona tsamaiso pafara e ka tsamaisa liphetoho tsohle.

PCR na le mehato e meraro: denaturation, selelekela annealing le elongation.

1. denaturation. Ka mocheso oa 94-95 likhato Celsius proshodit roba litlamo le hydrogen pakeng tsa liketane tse peli tsa DNA, 'me ka lebaka leo re fumana tse peli limolek'hule le' ngoe-tsielehile.

2. annealed primers. Ka mocheso oa likhato 50-60 centigrade le hlaha primers kamohelo ba lipheletsong tsa limolek'hule le 'ngoe-tsielehile nucleic acid e ho ea ka mofuta oa complementarity.

3. Elongation. Ka mocheso oa likhato tse 72 e tsa maiketsetso morali habeli tsielehile limolek'hule acid e deoxyribonucleic.

DNA hlahlamanya

Limolek'hule mekhoa ho etsa lipatlisiso tsa likokoana-hloko hangata o ne a hloka tsebo e ya tatelano ea nucleotides ka molek'hule ea acid e deoxyribonucleic. Ho fumana hore na khoutu liphatsa tsa lefutso di hlahlamantswe ho. tlhathoba limolek'hule tsa nakong e tlang li tla thehiloe tsebong e fumanoeng ka boikemisetso ba a latellana joang batho.

Latelang mefuta hlahlamanya:

• hlahlamanya ke Maxam-Gilbert;
• Sanger hlahlamanya;
• pyrosequencing;
• nanoporovoe hlahlamanya.