Gukuramo Acide Nucleic hamwe nuburyo bwa Magnetique

Intangiriro

Gukuramo Acide Nucleic ni iki?

Mu magambo yoroshye cyane, gukuramo aside nucleic ni ugukuraho RNA na / cyangwa ADN kuri sample hamwe nibirenga byose bidakenewe. Inzira yo gukuramo itandukanya acide nucleic ntangarugero kandi ikayitanga muburyo bwa eluate yibanze, itarangwamo imyanda nibihumanya bishobora kugira ingaruka kubisabwa byose.

Gukoresha Acide Nucleic Acide

Acide nucleic acide ikoreshwa mubwinshi bwibikorwa bitandukanye, bitandukanye mubikorwa bitandukanye. Ubuvuzi wenda ni agace gakoreshwa cyane, hamwe na RNA na ADN bisukuye bisabwa kugirango habeho intego zitandukanye zo kwipimisha.

Gusaba gukuramo aside nucleic mubuvuzi harimo:

- Kwiyongera kwa PCR na qPCR

- Urukurikirane rw'ibisekuru bizakurikiraho (NGS)

- Amplification ishingiye kuri SNP Genotyping

- Array ishingiye kuri Genotyping

- Kugaburira Enzyme Kurya

- Isesengura ukoresheje Guhindura Enzymes (urugero Ligation na Cloning)

Hariho nizindi nzego zirenze ubuvuzi aho hakoreshwa gukuramo aside nucleic, harimo ariko ntibigarukira kubipimisha ba se, forensic na genomics.

Amateka Mugufi yo Gukuramo Acide Nucleic

Gukuramo ADNAmatariki maremare, hamwe n’ubwigunge bwa mbere buzwi bwakozwe n’umuganga w’Ubusuwisi witwa Friedrich Miescher mu 1869. Miescher yari yizeye gukemura amahame remezo y’ubuzima agena imiterere y’imiterere ya selile. Amaze kunanirwa na lymphocytes, yashoboye kubona imvura idasanzwe ya ADN ivuye muri leucocytes iboneka muri pus ku gitambaro cyajugunywe. Yabikoze yongeramo aside hanyuma alkali muri selile kugirango asige cytoplazme ya selile, hanyuma akora protocole yo gutandukanya ADN nizindi poroteyine.

Nyuma yubushakashatsi bwakozwe na Miescher, abandi bahanga benshi bagiye gutera imbere no guteza imbere tekinike yo gutandukanya no kweza ADN. Edwin Joseph Cohn, umuhanga mu bya poroteyine yateje imbere uburyo bwinshi bwo kweza poroteyine mu gihe cya WW2. Yashinzwe gutandukanya serumu albumin agace ka plasma yamaraso, ifite akamaro mukubungabunga umuvuduko wa osmotic mumitsi yamaraso. Ibi byari ingenzi cyane kugirango abasirikari babeho.

Mu 1953, Francis Crick, hamwe na Rosalind Franklin na James Watson, bagennye imiterere ya ADN, berekana ko yari igizwe n'imirongo ibiri y'iminyururu miremire ya nucleotide acide nucleique. Ubu buvumbuzi bwavumbuye inzira Meselson na Stahl, bashoboye gukora protocole ya density gradient centrifugation protocole yo gutandukanya ADN na bagiteri E. Coli kuko bagaragaje ko ADN yigana igice cya kabiri mu bushakashatsi bwabo.

Ubuhanga bwo Gukuramo Acide Nucleic

Ni ibihe byiciro 4 byo gukuramo ADN?
Uburyo bwose bwo kuvoma butetse kugeza kuntambwe imwe yibanze.

Guhagarika Akagari. Iki cyiciro, kizwi kandi nka lysis selile, gikubiyemo gusenya urukuta rw'utugingo na / cyangwa ingirabuzimafatizo, kugirango urekure amazi yo mu nda arimo aside nucleic acide y'inyungu.

Gukuraho imyanda idakenewe. Ibi birimo lipide ya membrane, proteyine nizindi aside nucleique idakenewe ishobora kubangamira porogaramu zo hasi.

Kwigunga. Hariho uburyo butandukanye bwo gutandukanya acide nucleic acide yinyungu kuva lysate yahanaguwe wakoze, igwa hagati yibyiciro bibiri byingenzi: igisubizo gishingiye cyangwa gihamye (reba igice gikurikira).

Kwibanda. Acide nucleic aside imaze gutandukanywa nibindi byanduza byose hamwe nudukoko, bitangwa muri eluate yibanze cyane.

Ubwoko bubiri bwo gukuramo
Hariho ubwoko bubiri bwo gukuramo aside nucleic - uburyo bushingiye kubisubizo hamwe nuburyo bukomeye bwa leta. Uburyo bushingiye kubisubizo buzwi kandi nkuburyo bwo kuvoma imiti, kuko burimo gukoresha imiti yo kumena selile no kugera kubintu bya nucleic. Ibi birashobora kuba bikoresha ibinyabuzima nka fenol na chloroform, cyangwa bitangiza cyane bityo rero bikaba bisabwa cyane cyane ibinyabuzima nka Proteinase K cyangwa gelika ya silika.

Ingero zuburyo butandukanye bwo kuvoma imiti kugirango usenye selile harimo:

- Osmotic guturika kwa membrane

- Gusya kwa Enzymatique y'urukuta rw'akagari

- Solubilisation ya membrane

- Hamwe nogukoresha ibikoresho

- Hamwe no kuvura alkali

Ubuhanga bukomeye bwa leta, buzwi kandi nkuburyo bwubukanishi, burimo gukoresha uburyo ADN ikorana na substrate ikomeye. Muguhitamo isaro cyangwa molekile ADN izahuza ariko analyte ntabwo, birashoboka gutandukanya byombi. Ingero zubuhanga bukomeye bwo gukuramo harimo gukoresha silika na masaro.

Gukuramo Amasaro ya Magnetic Yasobanuwe

Uburyo bwo Gukuramo Amashanyarazi
Ubushobozi bwo gukuramo hakoreshejwe amasaro ya magneti bwamenyekanye bwa mbere muri patenti yo muri Amerika yatanzwe na Trevor Hawkins, ku kigo cy’ubushakashatsi cya Whitehead Institute. Iyi patenti yemeje ko bishoboka gukuramo ibikoresho bya genetike ubihambira ku kintu gikomeye gishobora kuba isaro. Ihame ni uko ukoresha isaro rya magnetique ikora cyane aho ibintu bya genetike bizahurira, hanyuma bigashobora gutandukanywa nindengakamere ukoresheje imbaraga za rukuruzi hanze yubwato bufite icyitegererezo.

Kuberiki Ukoresha Gukuramo Amashanyarazi?
Tekinoroji yo gukuramo amasaro ya magnetique iragenda igaragara cyane, bitewe nubushobozi ifite muburyo bwo kuvoma vuba kandi neza. Mu bihe byashize habaye iterambere ryamasaro ya magnetiki ikora cyane hamwe na sisitemu ya buffer ikwiye, yatumye bishoboka ko hakorwa aside nucleic acide hamwe nakazi ko gukora cyane kandi bitanga umusaruro. Nanone, uburyo bwo gukuramo amasaro ya magneti ntabwo bukubiyemo intambwe ya centrifugation ishobora gutera imbaraga zo gukata zisenya ibice birebire bya ADN. Ibi bivuze ko imirongo miremire ya ADN ikomeza kuba ntamakemwa, ifite akamaro mugupima genomics.