Intshayelelo
Yintoni iNucleic Acid Extraction?
Kwimiqathango elula kakhulu, i-nucleic acid extraction kukukhutshwa kwe-RNA kunye / okanye i-DNA kwisampuli kunye nayo yonke into engaphezulu engeyomfuneko. Inkqubo yokukhutshwa ihlukanisa i-nucleic acids kwisampulu kwaye ivelise ngendlela ye-eluate egxininisiweyo, ekhululekile kwi-diluents kunye neengcoliseko ezinokuchaphazela naziphi na izicelo ezisezantsi.
Ukusetyenziswa kweNucleic Acid Extraction
I-nucleic acids ecocekileyo isetyenziswa kwi-plethora yezicelo ezahlukeneyo, ukusuka kumashishini amaninzi ahlukeneyo. Ukhathalelo lwempilo mhlawumbi yeyona ndawo isetyenziswa kakhulu, kunye ne-RNA esulungekileyo kunye ne-DNA efunekayo kwintlaninge yeenjongo ezahlukeneyo zovavanyo.
Ukusetyenziswa kwe-nucleic acid extraction kukhathalelo lwezempilo kubandakanya:
-Ulandelelwano lwesiZukulwana esilandelayo (NGS)
-Amplification-based SNP Genotyping
-I-Array-based Genotyping
- Unyino lwe-Enzyme yokwetyisa
-Uhlalutya usebenzisa i-Enzymes yoHlengahlengiso (umzekelo, ukudibanisa kunye nokuKlonya)
Kukwakho neminye imimandla engaphaya kokhathalelo lwempilo apho kusetyenziswa i-nucleic acid extraction, kubandakanywa kodwa kungaphelelanga kuvavanyo lotata, i-forensics kunye ne-genomics.
Imbali emfutshane yeNucleic Acid yokutsalwa
Ukukhutshwa kwe-DNAyaqala kudala, kunye nokwahlukaniswa kokuqala okwaziwayo kwenziwa ngugqirha waseSwitzerland ogama linguFriedrich Miescher ngo-1869. UMiescher wayenethemba lokusombulula imigaqo-siseko yobomi ngokumisela ukwakheka kweekhemikhali zeeseli. Emva kokungaphumeleli kwi-lymphocytes, wakwazi ukufumana i-precipitate ekrwada ye-DNA evela kwi-leucocytes efunyenwe kwi-pus kwiibhandeji ezilahliweyo. Wakwenza oku ngokongeza iasidi kunye nealkali kwiseli ukushiya icytoplasm yeseli, waza waphuhlisa iprotocol yokwahlula iDNA kwezinye iiproteni.
Ukulandela uphando olwenziwa nguMiescher, ezinye izazinzulu ezininzi ziye zaqhubela phambili kwaye zaphuhlisa iindlela zokwahlula kunye nokucoca iDNA. U-Edwin Joseph Cohn, isazi seprotheni savelisa iindlela ezininzi zokuhlanjululwa kweeprotheyini ngexesha leWW2. Wayenoxanduva lokuhlukanisa i-serum albumin fraction ye-plasma yegazi, ebalulekileyo ekugcineni uxinzelelo lwe-osmotic kwimithambo yegazi. Oku kwakubalulekile ekugcineni amajoni ephila.
Ngowe-1953 uFrancis Crick, ekunye noRosalind Franklin noJames Watson, bafumanisa indlela iDNA eyakhiwe ngayo, nto leyo ebonisa ukuba yayiyilwe yimisonto emibini yemixokelelwane emide yenucleic acid nucleotides. Oku kufunyenweyo kwavula indlela kaMeselson kunye noStahl, abakwaziyo ukuphuhlisa iprotocol ye-gradient centrifugation protocol ukuba bahlukanise i-DNA kwiibhaktheriya ze-E. Coli njengoko babonisa ukuphindaphinda kwe-semi-conservative ye-DNA ngexesha lovavanyo lwabo lwe-1958.
Ubuchwephesha bokutsalwa kwe-Nucleic Acid
Ngawaphi amanqanaba ama-4 okukhutshwa kwe-DNA?
Zonke iindlela zokutsalwa ziwela kumanyathelo asisiseko afanayo.
Ukuphazamiseka kweSeli. Eli nqanaba, elikwabizwa ngokuba yi-cell lysis, libandakanya ukudiliza udonga lweseli kunye / okanye i-membrane yeseli, ukuze kukhululwe i-intra-cellular fluids equkethe i-nucleic acids enomdla.
Ukususwa kobutyobo obungafunwayo. Oku kubandakanya i-membrane lipids, iiprotheni kunye nezinye i-nucleic acids ezingafunekiyo ezinokuphazamisa izicelo ezisezantsi.
Ukubekwa wedwa. Kukho iindlela ezahlukeneyo zokuhlukanisa i-nucleic acids yomdla kwi-lysate ecocekileyo oyidalile, ewela phakathi kweendidi ezimbini eziphambili: isisombululo esisekelwe okanye esiqinileyo (jonga icandelo elilandelayo).
Ukugxila. Emva kokuba i-nucleic acids ibe yodwa kuzo zonke ezinye izinto ezingcolileyo kunye ne-diluents, zifakwe kwi-eluate egxininiswe kakhulu.
Iindidi ezimbini zokutsalwa
Kukho iintlobo ezimbini ze-nucleic acid extraction - iindlela ezisekelwe kwisisombululo kunye neendlela zombuso oqinileyo. Indlela esekelwe kwisisombululo yaziwa ngokuba yindlela yokukhupha imichiza, njengoko ibandakanya ukusebenzisa iikhemikhali ukudiliza iseli kunye nokufikelela kwizinto ze-nucleic. Oku kunokusebenzisa iikhompawundi eziphilayo ezifana nephenol kunye nekloroform, okanye ezona zinto zingeyongozi kangako kwaye ngoko ke iikhompawundi ezicetyiswayo ngakumbi njengeProteinase K okanye ijeli yesilica.
Imizekelo yeendlela ezahlukeneyo zokutsalwa kweekhemikhali zokophula iseli ziquka:
- Ukuqhekeka kwe-Osmotic ye-membrane
- Ukwetyisa i-Enzymatic yodonga lweseli
- I-Solubilisation ye-membrane
- Ngezokucoca
- Ngonyango lwe-alkali
Ubuchwephesha bobume obuqinileyo, obukwabizwa ngokuba ziindlela zoomatshini, kubandakanya ukuxhaphaza indlela i-DNA edibana ngayo ne-substrate eqinileyo. Ngokukhetha i-bead okanye i-molecule i-DNA iya kubophelela kuyo kodwa i-analyte ayifuni, kunokwenzeka ukuhlukanisa ezimbini. Imizekelo yeendlela eziqinileyo zokutsalwa kwesigaba esiquka ukusebenzisa isilica kunye namaso azibuthe.
Ukutsalwa kweMaso yeMagnetic kucacisiwe
Indlela yokutsalwa kweMaso yeMagnetic
Ukubanakho ukutsalwa kusetyenziswa amaso kazibuthe kwavunywa okokuqala kwi-patent yase-US efakwe nguTrevor Hawkins, kwiziko lophando le-Whitehead Institute. Le patent yavuma ukuba kwakunokwenzeka ukukhupha imathiriyeli yemfuza ngokuyibophelela kumthwali wenkxaso oqinileyo, onokuba liso lamagnetic. Umgaqo-siseko kukuba usebenzisa i-magnetic bead esebenza kakhulu apho i-genetic material iya kubophelela kuyo, enokuthi emva koko yahlulwe kwi-supernatant ngokusebenzisa amandla omagnetic ukuya ngaphandle kwenqanawa ebambe isampuli.
Kutheni Usebenzisa iMagnetic Bead Extraction?
Itekhnoloji yokutsalwa kweentsimbi zeMagnetic iya ixhaphaka kakhulu, ngenxa yesakhono esinayo kwiinkqubo zokutsalwa ezikhawulezayo nezisebenzayo. Kutshanje kuye kwakho uphuhliso lwamaso kazibuthe asebenza kakhulu aneenkqubo ezifanelekileyo ze-buffer, ezenze i-automation ye-nucleic acid extraction kunye nokuhamba komsebenzi okukhanya kakhulu kunye neendleko ezifanelekileyo. Kwakhona, iindlela zokutsalwa kwamaso ezizibuthe azibandakanyi amanyathelo okubeka kucentrifugation anokubangela amandla okucheba aqhekeze amaqhekeza amade eDNA. Oku kuthetha ukuba imicu emide ye-DNA ihlala injalo, nto leyo ibalulekileyo kuvavanyo lwe-genomics.
Ixesha lokuposa: Nov-25-2022