Isingeniso
Iyini i-Nucleic Acid Extraction?
Ngamagama alula kakhulu, ukukhishwa kwe-nucleic acid ukususwa kwe-RNA kanye/noma i-DNA kusampula nakho konke okweqile okungadingekile.Inqubo yokukhipha ihlukanisa ama-nucleic acid kusampula futhi iwakhiphe ngendlela ye-eliate egxilile, engenazo izincibilikisi nokungcola okungase kuthinte noma yiziphi izinhlelo zokusebenza ezansi nomfula.
Izicelo ze-Nucleic Acid Extraction
Ama-nucleic acid ahlanzekile asetshenziswa ezinhlobonhlobo zezinhlelo zokusebenza ezihlukene, kusukela ezimbonini eziningi ezahlukene.Ukunakekelwa kwezempilo mhlawumbe indawo lapho kusetshenziswa khona kakhulu, nge-RNA ehlanzekile kanye ne-DNA edingekayo ngenqwaba yezinjongo ezihlukene zokuhlola.
Izicelo zokukhishwa kwe-nucleic acid ekunakekelweni kwezempilo zifaka:
- I-Next Generation Sequencing (NGS)
- I-Amplification-based SNP Genotyping
- I-Array-based Genotyping
- Ukuvinjelwa kwe-Enzyme Ukugaya
- Ihlaziya kusetshenziswa ama-Enzymes ashintshayo (isb. Ukuhlanganisa nokuhlanganisa)
Kukhona nezinye izinkambu ngale kokunakekelwa kwezempilo lapho kusetshenziswa khona i-nucleic acid, okuhlanganisa kodwa kungagcini ekuhlolweni kobaba, i-forensics kanye ne-genomics.
Umlando Omfishane We-Nucleic Acid Extraction
Ukukhishwa kwe-DNAisukela kudala, nokuhlukaniswa kokuqala okwaziwayo kwenziwa udokotela waseSwitzerland ogama lakhe linguFriedrich Miescher ngo-1869. UMiescher wayenethemba lokuxazulula izimiso eziyisisekelo zokuphila ngokunquma ukwakheka kwamakhemikhali kwamangqamuzana.Ngemva kokwehluleka ngama-lymphocyte, wakwazi ukuthola imvula engahluziwe ye-DNA kumaleukocyte atholakala obovu kumabhandeshi alahliwe.Lokhu wakwenza ngokufaka i-asidi bese kuba i-alkali engqamuzaneni ukuze ashiye i-cytoplasm yeseli, wabe esesungula isimiso somthetho sokuhlukanisa i-DNA kwamanye amaprotheni.
Ngemva kocwaningo luka-Miescher oluyinselele, abanye ososayensi abaningi baye baqhubekela phambili futhi bathuthukisa amasu okuhlukanisa nokuhlanza i-DNA.U-Edwin Joseph Cohn, usosayensi wamaprotheni wakha amasu amaningi okuhlanza amaprotheni phakathi ne-WW2.Wayenomthwalo wemfanelo wokuhlukanisa ingxenye ye-albumin ye-serum ye-plasma yegazi, ebalulekile ekugcineni umfutho we-osmotic emithanjeni yegazi.Lokhu bekubalulekile ekugcineni amasosha ephila.
Ngo-1953 uFrancis Crick, kanye noRosalind Franklin noJames Watson, banquma ukwakheka kwe-DNA, okubonisa ukuthi yayakhiwe imicu emibili yamaketanga amade e-nucleic acid nucleotide.Lokhu kutholakala okuyimpumelelo kwavulela u-Meselson no-Stahl indlela, abakwazile ukwenza iphrothokholi ye-gradient centrifugation protocol ukuze bahlukanise i-DNA kubhaktheriya ye-E. Coli njengoba babonisa ukuphindaphinda kwe-DNA e-semi-conservative ngesikhathi sokuhlola kwabo kwango-1958.
Izindlela Zokukhipha I-Nucleic Acid
Yiziphi izigaba ezi-4 zokukhishwa kwe-DNA?
Zonke izindlela zokukhipha zifinyelela ezinyathelweni ezifanayo eziyisisekelo.
Ukuphazamiseka Kweseli.Lesi sigaba, esaziwa nangokuthi i-cell lysis, sifaka ukubhidliza udonga lweseli kanye/noma ulwelwesi lweseli, ukuze kukhululwe uketshezi olungaphakathi kwamaselula oluqukethe ama-nucleic acid athandwayo.
Ukususwa Kwemfucumfucu Engadingeki.Lokhu kuhlanganisa i-membrane lipids, amaprotheni namanye ama-nucleic acid angadingeki angaphazamisa ukusebenza komfula.
Ukuzihlukanisa.Kunezindlela eziningana ezihlukene zokuhlukanisa ama-nucleic acid anentshisekelo ku-lysate esuliwe oyidalile, ewela phakathi kwezigaba ezimbili eziyinhloko: isimo esisekelwe kwisixazululo noma esiqinile (bona isigaba esilandelayo).
Ukugxila.Ngemuva kokuthi ama-nucleic acid ehlukanisiwe kuzo zonke ezinye izinto ezingcolile kanye nama-diluents, ethulwa ngendlela egxilile kakhulu.
Izinhlobo Ezimbili Zokukhipha
Kunezinhlobo ezimbili zokukhishwa kwe-nucleic acid - izindlela ezisekelwe kwisisombululo nezindlela zesimo esiqinile.Indlela esekelwe kwisixazululo yaziwa nangokuthi indlela yokukhipha amakhemikhali, njengoba ihilela ukusebenzisa amakhemikhali ukudiliza iseli nokufinyelela ku-nucleic material.Lokhu kungaba ukusebenzisa izinhlanganisela eziphilayo ezifana ne-phenol ne-chloroform, noma izinhlanganisela ezingaphili kangako futhi ezituswa kakhulu njenge-Proteinase K noma ijeli ye-silica.
Izibonelo zezindlela ezihlukene zokukhipha amakhemikhali zokudiliza iseli zifaka:
- Ukuqhekeka kwe-Osmotic ye-membrane
- Ukugaya kwe-Enzymatic kodonga lwamaseli
- I-Solubilisation ye-membrane
- Ngezokuhlanza
- Ngokwelashwa kwe-alkali
Amasu ombuso oqinile, owaziwa nangokuthi izindlela zemishini, afaka ukuxhaphaza ukuthi i-DNA ixhumana kanjani ne-substrate eqinile.Ngokukhetha ubuhlalu noma i-molecule i-DNA ezobophezela kuyo kodwa i-analyte ingavumi, kungenzeka ukuhlukanisa kokubili.Izibonelo zamasu okukhipha isigaba esiqinile okuhlanganisa ukusebenzisa i-silica nobuhlalu obuzibuthe.
Isizinda Sobuhlalu Bubukhazikhazi Kuyachazwa
I-Magnetic Bead Extraction Method
Amandla okukhishwa kusetshenziswa ubuhlalu kazibuthe aqashelwa okokuqala egunyazweni lase-US elifakwe ngu-Trevor Hawkins, wesikhungo socwaningo se-Whitehead Institute.Le lungelo lobunikazi yavuma ukuthi kwakungenzeka ukukhipha izakhi zofuzo ngokuzibophezela esiphathini esiqinile sokusekela, okungaba ubuhlalu obuzibuthe.Isimiso siwukuthi usebenzisa ubuhlalu obukazibuthe obusebenza kahle kakhulu lapho okofuzo kuzobophezela kubo, okungabe sekuhlukaniswa namandla amakhulu ngokusebenzisa amandla kazibuthe ngaphandle komkhumbi obambe isampula.
Kungani Usebenzisa I-Magnetic Bead Extraction?
Ubuchwepheshe bokukhipha ubuhlalu obuzibuthe buya ngokuya budlanga, ngenxa yamandla obunawo ezinqubo zokukhipha ngokushesha nangempumelelo.Ezikhathini zamuva nje kube nentuthuko yobuhlalu obuzibuthe obusebenza kahle kakhulu obunamasistimu afanele we-buffer, okwenze kube nokwenzeka ukuzenzekelayo kokukhipha i-nucleic acid kanye nokugeleza komsebenzi okukhanya kakhulu kwensiza futhi ukonga.Futhi, izindlela zokukhipha ubuhlalu kazibuthe azibandakanyi izinyathelo zokubeka i-centrifugation ezingabangela amandla okugunda ahlephula izingcezu ezinde ze-DNA.Lokhu kusho ukuthi imicu emide ye-DNA ihlala injalo, okubalulekile ekuhlolweni kwe-genomics.
Isikhathi sokuthumela: Nov-25-2022