Nucleic Acid Extraction thiab Magnetic Bead Method

Taw qhia

Nucleic Acid Extraction yog dab tsi?

Hauv cov ntsiab lus yooj yim tshaj plaws, nucleic acid extraction yog tshem tawm ntawm RNA thiab / lossis DNA los ntawm cov qauv thiab tag nrho cov ntau dhau uas tsis tsim nyog.Cov txheej txheem ntawm extraction cais cov nucleic acids los ntawm ib tug qauv thiab yields lawv nyob rau hauv daim ntawv ntawm ib tug concentrated eluate, dawb los ntawm diluents thiab contaminants uas yuav cuam tshuam rau ib tug downstream daim ntaub ntawv.

Kev siv ntawm Nucleic Acid Extraction

Purified nucleic acids yog siv nyob rau hauv plethora ntawm ntau daim ntawv thov, xws li nyob rau hauv ntau yam kev lag luam.Kev kho mob yog kab tias thaj chaw uas nws siv feem ntau, nrog rau RNA thiab DNA kom huv si xav tau rau ntau lub hom phiaj sib txawv.

Kev siv ntawm nucleic acid extraction hauv kev kho mob suav nrog:

- PCR thiab qPCR Amplification

- Next Generation Sequencing (NGS)

- Amplification-based SNP Genotyping

- Array-based Genotyping

- Kev txwv enzyme digestion

- Kev tshuaj xyuas siv Modifying Enzymes (xws li Ligation thiab Cloning)

Kuj tseem muaj lwm qhov chaw tshaj li kev kho mob uas siv cov nucleic acid extraction, suav nrog tab sis tsis txwv rau kev kuaj niam txiv, forensics thiab genomics.

Keeb kwm luv luv ntawm Nucleic Acid Extraction

DNA rho tawmhnub rov qab mus ntev, nrog thawj zaug paub kev cais tawm tau ua los ntawm ib tus kws kho mob Swiss hu ua Friedrich Miescher hauv xyoo 1869. Miescher vam tias yuav daws cov ntsiab lus tseem ceeb ntawm lub neej los ntawm kev txiav txim siab cov tshuaj muaj pes tsawg leeg ntawm cov hlwb.Tom qab ua tsis tiav nrog lymphocytes, nws muaj peev xwm tau txais ib qho av nplaum ntawm DNA los ntawm leukocytes pom hauv cov kua txiv ntawm cov ntaub qhwv pov tseg.Nws ua li no los ntawm kev ntxiv cov kua qaub thiab tom qab ntawd alkali rau hauv lub cell kom tawm ntawm lub cell lub cytoplasm, thiab tom qab ntawd tsim ib txoj cai los cais cov DNA ntawm lwm cov proteins.

Ua raws li Miescher txoj kev tshawb fawb hauv av, ntau lwm tus kws tshawb fawb tau mus ua ntej thiab tsim cov tswv yim los cais thiab ntxuav DNA.Edwin Joseph Cohn, tus kws tshawb fawb txog protein ntau tau tsim ntau cov tswv yim rau kev ntxuav cov protein thaum WW2.Nws yog lub luag haujlwm rau kev cais cov serum albumin feem ntawm cov ntshav ntshav, uas yog qhov tseem ceeb hauv kev tswj hwm osmotic siab hauv cov hlab ntsha.Qhov no yog qhov tseem ceeb rau kev ua kom cov tub rog ciaj sia.

Xyoo 1953 Francis Crick, nrog rau Rosalind Franklin thiab James Watson, tau txiav txim siab cov qauv ntawm DNA, qhia tias nws tau tsim los ntawm ob txoj hlua ntev ntawm nucleic acid nucleotides.Qhov kev tshawb fawb no tau txiav txoj kev rau Meselson thiab Stahl, uas muaj peev xwm los txhim kho DNI-corriater suplication ntawm DNA thaum lawv 1958 sim.

Cov txheej txheem ntawm Nucleic Acid Extraction

Dab tsi yog 4 theem ntawm DNA rho tawm?
Tag nrho cov txheej txheem extraction boil mus rau tib theem pib.

Cell cuam tshuam.Cov theem no, tseem hu ua cell lysis, suav nrog kev rhuav tshem cov phab ntsa ntawm tes thiab / lossis cov cell membrane, thiaj li tso cov kua dej hauv cellular uas muaj cov nucleic acids txaus siab.

Kev tshem tawm cov khib nyiab uas tsis xav tau.Qhov no suav nrog daim nyias nyias lipid, cov proteins thiab lwm yam tsis xav tau nucleic acids uas tuaj yeem cuam tshuam rau kev siv qis qis.

Muab cais nyob ib leeg.Muaj ntau txoj hauv kev los cais cov nucleic acids ntawm kev txaus siab los ntawm kev tshem tawm lysate uas koj tsim, uas poob ntawm ob pawg tseem ceeb: kev daws teeb meem los yog lub xeev ruaj khov (saib ntu tom ntej).

Nco ntsoov.Tom qab cov nucleic acids tau raug cais tawm los ntawm tag nrho lwm cov kab mob thiab cov diluents, lawv tau nthuav tawm hauv cov ntsiab lus siab heev.

Ob hom Extraction
Muaj ob hom nucleic acid extraction - daws raws li txoj kev thiab cov txheej txheem hauv lub xeev.Txoj kev daws teeb meem raws li txoj kev tseem hu ua chemical extraction method, raws li nws koom nrog kev siv tshuaj los rhuav tshem ntawm tes thiab nkag mus rau cov khoom siv nucleic.Qhov no tuaj yeem siv cov organic tebchaw xws li phenol thiab chloroform, lossis tsawg dua kev puas tsuaj thiab yog li kev pom zoo ntau dua cov tshuaj inorganic xws li Proteinase K lossis silica gel.

Piv txwv ntawm kev sib txawv ntawm cov tshuaj rho tawm los rhuav tshem lub cell muaj xws li:

- Osmotic rupturing ntawm daim nyias nyias

- Enzymatic digestion ntawm cell phab ntsa

- Solubilization ntawm daim nyias nyias

- Nrog detergents

- Nrog kev kho alkali

Cov txheej txheem hauv lub xeev, tseem hu ua cov txheej txheem siv tshuab, suav nrog kev siv DNA cuam tshuam li cas nrog cov khoom siv substrate.Los ntawm kev xaiv ib lub hlaws lossis molecule uas DNA yuav khi rau tab sis qhov ntsuas tsis tau, nws muaj peev xwm cais ob.Piv txwv ntawm cov txheej txheem kev rho tawm cov txheej txheem suav nrog kev siv silica thiab cov hlaws sib nqus.

Magnetic Bead Extraction piav qhia

Magnetic Bead Extraction Method
Lub peev xwm rau kev rho tawm siv cov hlaws sib nqus tau pom thawj zaug hauv Teb Chaws Asmeskas patent thov los ntawm Trevor Hawkins, rau lub koom haum tshawb fawb Whitehead.Qhov no patent tau lees paub tias nws muaj peev xwm rho tawm cov khoom siv noob caj noob ces los ntawm kev khi lawv mus rau cov khoom siv txhawb nqa, uas tuaj yeem yog cov hlaws sib nqus.Lub hauv paus ntsiab lus yog tias koj siv cov hlaws sib nqus ua haujlwm zoo heev rau cov khoom siv caj ces yuav khi rau, uas tuaj yeem raug cais tawm ntawm cov supernatant los ntawm kev siv lub zog sib nqus mus rau sab nraud ntawm lub nkoj tuav cov qauv.

Vim li cas thiaj siv Magnetic Bead Extraction?
Sib nqus hlaws extraction tshuab tau dhau los ua ntau dua, vim tias nws muaj peev xwm tuav tau cov txheej txheem rho tawm sai thiab ua tau zoo.Nyob rau lub sijhawm tsis ntev los no tau muaj kev txhim kho ntawm cov hlaws zoo sib nqus ua haujlwm nrog cov tshuab tsis zoo, uas tau ua kom muaj peev xwm ua tau zoo ntawm nucleic acid extraction thiab kev ua haujlwm uas muaj peev xwm ua tau zoo thiab tus nqi qis.Tsis tas li ntawd, cov txheej txheem sib nqus cov hlaws rho tawm tsis cuam tshuam nrog cov kauj ruam centrifugation uas tuaj yeem ua rau lub zog shear uas rhuav tshem cov DNA ntev dua.Qhov no txhais tau hais tias ntev strands ntawm DNA nyob twj ywm, uas yog ib qho tseem ceeb hauv kev ntsuam xyuas genomics.