Nhanganyaya
Chii chinonzi Nucleic Acid Extraction?
Mumashoko akareruka zvikuru, nucleic acid extraction ndiko kubviswa kweRNA uye/kana DNA kubva mumuenzaniso uye zvose zvakawandisa zvisingadiwi. Iyo nzira yekubvisa inoparadzanisa nucleic acids kubva mumuenzaniso uye inoaburitsa muchimiro cheyakazara eluate, isina kubva kune diluent uye zvinosvibisa zvinogona kukanganisa chero yakadzika maapplication.
Zvishandiso zveNucleic Acid Extraction
Yakanatswa nucleic acids inoshandiswa muhuwandu hwemashandisirwo akasiyana, kubva kune akawanda akasiyana maindasitiri. Hutano hunogona kunge ndiyo nharaunda yainonyanya kushandiswa, ine yakacheneswa RNA neDNA inodiwa kune dzakawanda dzakasiyana dzakasiyana dzekuyedza zvinangwa.
Zvishandiso zve nucleic acid kudhirowa mune hutano zvinosanganisira:
-Inotevera Generation Sequencing (NGS)
- Amplification-yakavakirwa SNP Genotyping
- Array-based Genotyping
- Kurambidza Enzyme Digestion
-Ongorora uchishandisa Kugadzirisa Enzymes (semuenzaniso Ligation uye Cloning)
Kune zvakare mamwe minda kunze kwehutano hwehutano uko kunoshandiswa nucleic acid kudhirowa, kusanganisira asi isina kuganhurirwa kune paternity kuyedzwa, forensics uye genomics.
Nhoroondo Pfupi yeNucleic Acid Kubviswa
Kutorwa kweDNAyakatanga kure, nekuzviparadzanisa nevamwe kwekutanga kwakaitwa nachiremba wekuSwitzerland ainzi Friedrich Miescher muna 1869. Miescher aitarisira kugadzirisa misimboti yakakosha yehupenyu nekuona kuumbwa kwemakemikari emaseru. Mushure mekukundikana ne lymphocytes, akakwanisa kuwana crude precipitate yeDNA kubva ku leukocyte inowanikwa mupus pamabhandeji akaraswa. Akaita izvi nekuwedzera asidhi uye ipapo alkali kune sero kusiya cytoplasm yesero, uye ndokugadzira chirongwa chekuparadzanisa DNA kubva kune mamwe mapuroteni.
Kutevera tsvakiridzo yepasi-pasi yaMiescher, mamwe masayendisiti mazhinji akaenderera mberi nekugadzira matekiniki ekuparadzanisa uye kuchenesa DNA. Edwin Joseph Cohn, musayendisiti weprotein akagadzira nzira dzakawanda dzekuchenesa mapuroteni panguva yeWW2. Aive nebasa rekuparadzanisa serum albumin chikamu cheplasma yeropa, iyo yakakosha mukuchengetedza osmotic pressure mutsinga dzeropa. Izvi zvaikosha kuti varwi varambe vari vapenyu.
Muna 1953 Francis Crick, pamwe naRosalind Franklin naJames Watson, vakasarudza maumbirwo eDNA, achiratidza kuti yakanga yakaumbwa netambo mbiri dzecheni refu dze nucleic acid nucleotides. Kuwanikwa kwebudiriro uku kwakagadzirira nzira kuna Meselson naStahl, vakakwanisa kugadzira density gradient centrifugation protocol kuparadzanisa DNA kubva kuE. Coli mabhakitiriya pavakaratidza kudzokororwa kwesemi-Conservative yeDNA panguva yavo yekuyedza 1958.
Matekiniki eNucleic Acid Kubvisa
Ndeapi matanho mana ekutorwa kweDNA?
Nzira dzese dzekuchera dzinodzika kusvika kumatanho akakosha akafanana.
Kukanganiswa kweSero. Danho iri, rinozivikanwawo sero sero lysis, rinosanganisira kuputsa rusvingo rwesero uye/kana membrane yesero, kuitira kuti isunungure intra-cellular fluid ine nucleic acids inofarira.
Kubviswa Kwemarara Asingadiwi. Izvi zvinosanganisira membrane lipids, mapuroteni uye mamwe asingadiwe nucleic acids ayo anogona kukanganisa neakadzika maapplication.
Kuiswa kwemurwere kwake ega. Pane nzira dzinoverengeka dzakasiyana dzekuparadzanisa nucleic acids yekufarira kubva kune yakacheneswa lysate yawakagadzira, iyo inowira pakati pemapoka makuru maviri: mhinduro yakavakirwa kana yakasimba mamiriro (ona chikamu chinotevera).
Concentration. Mushure mokunge nucleic acids yave yakaparadzaniswa kubva kune zvimwe zvose zvinosvibisa uye diluents, inoratidzwa mune yakanyanyisa-yakanyanyisa eluate.
Mhando mbiri dzekubvisa
Kune marudzi maviri eiyo nucleic acid yekubvisa - mhinduro yakavakirwa nzira uye yakasimba nyika nzira. Iyo mhinduro yakavakirwa nzira inozivikanwawo seyo nzira yekubvisa makemikari, sezvo inosanganisira kushandisa makemikari kuputsa sero uye kuwana iyo nucleic zvinhu. Izvi zvinogona kunge zvichishandisa organic compounds senge phenol nechloroform, kana zvisinganyanye kukuvadza uye nekudaro zvinokurudzirwa inorganic compounds seProteinase K kana silica gel.
Mienzaniso yemhando dzakasiyana dzekubvisa makemikari nzira dzekupwanya sero dzinosanganisira:
- Osmotic kuputika kwe membrane
- Enzymatic digestion ye cell wall
- Solubilisation ye membrane
- Ne detergents
- Ne alkali kurapwa
Solid state matekiniki, anozivikanwawo sema mechanical nzira, anosanganisira kushandisa mabatiro anoita DNA ne substrate yakasimba. Nekusarudza bead kana molecule iyo DNA inosungirira pairi asi muongorori haaite, zvinokwanisika kupatsanura zviviri izvi. Mienzaniso yemaitiro akasimba-chikamu chekubvisa anosanganisira kushandisa silica uye magineti bead.
Magnetic Bead Extraction Inotsanangurwa
Iyo Magnetic Bead Extraction Method
Iko kugona kudhirowa uchishandisa magineti bead kwakatanga kuzivikanwa muUS patent yakaiswa naTrevor Hawkins, yeWhitehead Institute research institution. Iyi patent yakabvuma kuti zvaigoneka kuburitsa genetic zvinhu nekuzvisunga kune yakasimba tsigiro inotakura, inogona kunge iri magineti bead. Nheyo yacho ndeyekuti iwe unoshandisa yakanyanya functionalised magineti bead pairi iyo genetic material inosungirira pairi, iyo inogona kubva yapatsanurwa kubva kune supernatant nekuisa simba remagineti kunze kwemudziyo wakabata sample.
Sei Uchishandisa Magnetic Bead Extraction?
Magnetic bead yekubvisa tekinoroji iri kuramba ichiwanda, nekuda kwekugona kwairi kuita nekukurumidza uye nekuita maitiro ekubvisa. Munguva pfupi yapfuura kwave nekuvandudzwa kweanoshanda magineti mabedhi ane akakodzera mabuffer masisitimu, ayo akaita kuti otomatiki yeadhini nucleic acid ibviswe uye kufambiswa kwebasa kunove kwakanyanya zviwanikwa uye zvinodhura. Zvakare, nzira dzemagineti dzekubvisa bead hadzisanganisi nhanho dzecentrifugation dzinogona kukonzera masimba ekugera ayo anopwanya zvidimbu zveDNA. Izvi zvinoreva kuti tambo refu dzeDNA dzinoramba dzakasimba, izvo zvakakosha pakuongorora genomics.
Nguva yekutumira: Nov-25-2022