Ukuqalisa
Kuyini ukukhishwa kwe-nucleic acid?
Emigabeni elula kakhulu yemigomo, ukukhishwa kwe-nucleic acid ukususwa kwe-RNA kanye / noma i-DNA kusuka kusampula nakho konke okweqile okungadingekile. Inqubo yokukhishwa ihlukanisa ama-nucleic acid kusuka kusampula futhi ibanikele ngesimo se-exterent egxilile, ngaphandle kwezimali ezingcolisayo kanye nokungcola okungathinta noma yiziphi izinhlelo zokusebenza ezansi.
Izicelo zokukhishwa kwe-nucleic acid
Ama-nucleic acid ahlanzekile asetshenziswa ku-plethora yezinhlelo zokusebenza ezahlukahlukene, ezisukela ezimbonini eziningi ezahlukahlukene. Ukunakekelwa kwezempilo mhlawumbe yindawo lapho isetshenziswa khona kakhulu, nge-RNA ehlanziwe ne-DNA edingekayo kwinhloso ehlukile yezinjongo ezahlukahlukene zokuhlola.
Izicelo zokukhishwa kwe-nucleic acid ekunakekelweni kwezempilo zifaka:
- Ukulandelana kwesizukulwane esilandelayo (ngs)
- I-amplification-based snp genotyping
- I-genoty-based genotyping
- Imikhawulo yokugayeka kwe-enzyme
- Ukuhlaziya usebenzisa ukuguqula ama-enzyme (isib. I-ligation kanye nokuqina)
Kukhona nezinye izinkambu ezingaphezu kwezempilo lapho kusetshenziswa khona ukukhishwa kwezempilo lapho kusetshenziswa khona ukukhishwa kwe-nucleic acid, kufaka phakathi noma kungagcini ekuhlolweni kobaba, ama-forensics kanye ne-genomics.
Umlando omfishane wokukhishwa kwe-nucleic acid
Ukukhishwa kwe-DNAUkubuyela emuva kude, nokwahlukaniswa kokuqala kwaziwa kwenziwe ngudokotela waseSwitzerland ogama lakhe linguFriedrich Miescher ngo-1869. UMiescher ubenethemba lokuxazulula ukwakheka kwamakhemikhali kwamaseli. Ngemuva kokuhluleka ngama-lymphocyte, wakwazi ukuthola i-dresite ye-DNA eqondile evela kuma-leucocytes atholakala ku-pus kumabhandeshi alahliwe. Lokhu wakwenza ngokungeza i-acid bese u-alkali esitokisini ukushiya i-cytoplasm yeseli, wabe esethuthuka umthetho wokuhlukanisa i-DNA kwamanye amaprotheni.
Ngemuva kokucwaninga komhlaba okuphula umthetho we-Miescher, abanye ososayensi abaningi baye badlulela phambili futhi bathuthukise amasu ukuhlukanisa futhi bahlanze i-DNA. U-Edwin Joseph Cohn, usosayensi wamaprotheni wathuthukisa amasu amaningi ekuhlanjululweni kwamaprotheni ngesikhathi se-WW2. Ubephethe icala lokuhlukanisa ingxenyana ye-serum albhamuin ye-plasma yegazi, okubalulekile ekugcineni ingcindezi ye-osmotic emithanjeni yegazi. Lokhu bekubalulekile ukugcina amasosha ephila.
Ngo-1953 uFrancis Crick, kanye noRosalind Franklin noJames Watson, banquma ukwakheka kwe-DNA, bekhombisa ukuthi yenziwa imicu emibili yamaketanga amade we-nucleic acid nucleotides. Lokhu okutholwe yi-Precrough kudalulwe indlela kaMesonson noStahl, abakwaziyo ukuthuthukisa i-protocolol ye-gradity gradient centrity ukuhlukanisa i-DNA kusuka ku-E. Coli bacterium njengoba bekhombisa ukuphindaphindwa kwe-DNA ngesikhathi sabo sika-1958.
Amasu wokukhishwa kwe-nucleic acid
Yiziphi izigaba ezi-4 zokukhishwa kwe-DNA?
Zonke izindlela zokukhishwa zivulisa izinyathelo ezifanayo eziyisisekelo.
Ukuphazamiseka kweseli. Lesi sigaba, esaziwa nangokuthi i-Cell Lysis, sibandakanya ukudiliza udonga lweseli kanye / noma ulwelwesi lwamaseli, ukudedela uketshezi lwamaselula aqukethe ama-acid ama-nucleic anentshisekelo.
Ukususwa kwemfucumfucu engafuneki. Lokhu kufaka phakathi ama-membrane lipids, amaprotheni namanye ama-nucleic acid angafuneki angaphazamisa izinhlelo zokusebenza eziphansi.
Ukuzihlukanisa. Kunezindlela ezimbalwa ezahlukahlukene zokuhlukanisa ama-nucleic acid ezithakazelo kusuka ku-lysate esuliwe oyidalile, ewa phakathi kwezigaba ezimbili eziphambili: isixazululo esisekelwe esisekelweni noma esiqinile (bona isigaba esilandelayo).
Ukugxila. Ngemuva kokuthi ama-acid ama-nucleic ahlukaniswe nabo bonke abanye abangcoliso kanye nezinto ezingcolile, zivezwa eqenjini eligxile kakhulu.
Izinhlobo ezimbili zokukhishwa
Kunezinhlobo ezimbili zokukhishwa kwe-nucleic acid - izindlela ezisuselwa kusisombululo nezindlela eziqinile zombuso. Indlela yokususelwa kwesixazululo yaziwa nangokuthi indlela yokukhishwa kwamakhemikhali, ngoba ifaka ukusebenzisa amakhemikhali ukudiliza iseli futhi ufinyelele ezintweni ze-nucleic. Lokhu kungasebenzisa ama-organic arganic afana ne-phenol ne-chloroform, noma okulimazayo okunengozi ngakho-ke izincomo ze-inorganic ezinconyiwe ezinjenge-proteininase k noma i-silica gel.
Izibonelo zezindlela ezahlukahlukene zokukhishwa kwamakhemikhali ukuze zihlukanise iseli zifaka phakathi:
- Ukwephulwa kwe-osmotic of membrane
- Ukugayeka kwe-enzymatic kodonga lweseli
- Ukuhlukaniswa kolwelwesi
- ngama-detegents
- Ngokwelashwa kwe-alkali
Amasu wombuso aqinile, owaziwa nangokuthi izindlela zemishini, kubandakanya ukuxhaphaza ukuthi i-DNA ixhumana kanjani ne-substrate eqinile. Ngokukhetha ubuhlalu noma i-molecule okuyi-DNA ezobopha kuyo kepha i-analyte ngeke, kungenzeka ukuhlukanisa lokhu okubili. Izibonelo zamasu wokukhipha esigabeni esiqinile kubandakanya ukusebenzisa iSilica kanye nobuhlalu bakaMagnetic.
Kuchazwe isizinda sobude be-Magnetic
Indlela ye-Magnetic Bead Extuaction indlela
Amathuba okukhishwa asebenzisa ubuhlalu bamazibuthe okokuqala wamukelwa kumalungelo obunikazi ase-US afakwe yiTrevor Hawkent, kwiSikhungo Sokucwaninga ngesikhungo sase-Whitehead Institute. Le patent yavuma ukuthi kungenzeka ukuthi kwakhipha izinto zofuzo ngokuzibopha kumthwali oqinile wokuxhasa, ongaba ubuhlalu obubukhali. Umgomo ukuthi usebenzise i-Magnetic Bead esetshenziswa kakhulu lapho impahla yofuzo ezobopha khona, engahlukaniswa ne-supernatan ngokusebenzisa amandla kazibuthe ngaphandle komkhumbi obambe isampula.
Kungani usebenzise i-Magnetic Bead Extraction?
Ubuchwepheshe bokukhishwa kwe-magnetic bead buya ngokuya buvame kakhulu, ngenxa yamandla aphethwe yinqubo yokukhishwa okusheshayo neyokusebenza. Ezikhathini zamuva kube nokuthuthuka kobuhlalu obusebenza ngobukhulu obusebenza kahle ngezinhlelo ezifanele ze-buffer, ezenze ezenzakalelayo zokukhishwa kwe-nucleic acid kanye nokuhamba komsebenzi okukhanya kakhulu nokubi. Futhi, izindlela zokukhishwa kwe-kazibuthe zobuhlalu azihileli izinyathelo ze-centrifugation ezingadala amandla okugula aphula izingcezu ze-DNA ezinde ze-DNA. Lokhu kusho ukuthi imicu emide ye-DNA ihlala iqinile, okubalulekile ekuhlolweni kwe-genomics.
Isikhathi sePosi: Nov-25-2022