Nucleic Acid Extraction Sahil

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    NUCLEIC ACID EXTRACTION

    SAHIL KULKARNI

    SENIOR RESEARCH FELLOW

    HAFFKINE INSTITUTE

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    Where is DNA present inthe cell????

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    Purpose

    yTo releasenucleic acid from the cell for

    use in otherprocedures

    yMust be free from contamination with

    protein, carbohydrate,lipids orother

    nu

    cleic acids.yUsed purenucleic acids fortesting.

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    Isolation

    y Routinely isolated from human, fungal, bacterial andviral sources.

    y Pre treat to make nucleated cells available,

    y whole blood

    y Tissue samples

    y Microorganisms

    y Need sufficient sample for adequate yield.

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    Therearethree basic and one optional steps ina

    DNA/RNA extraction:Breakingthe cells open, commonlyreferred to as cell

    disruption orcelllysis,to exposethe DNA/RNA within. This is

    commonlyachieved bygrinding orsonicating the sample.

    Removing membranelipids byaddinga detergent.

    Removingproteins byaddingaprotease (optional butalmost

    always done).

    Precipitatingthe DNA/RNA withanalcohol usually ice-

    cold ethanol orisopropanol. Since DNA/RNA is insoluble inthese

    alcohols, it willaggregatetogether,givinga pelletupon

    centrifugation. This step also removes alcohol-soluble salt.

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    Two majortypes of methods :

    yPhenol-chloroform extraction

    yColumn purification

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    It is a liquid-liquid extractiontechnique

    inbiochemistry.

    Liquid-liquid extraction, also known

    as solvent extraction and partitioning,is a method to separate compoundsbased on their relative solubilities intwo different immiscible liquids. It isan extraction of a substance from one

    liquid phase into another liquid phase.It is widelyused in molecularbiology for

    isolating DNA, RNA and protein.

    Equalvolumes ofa phenol: chloroform

    mixtureand anaqueous sampleare mixed,

    formingabiphasic mixture.

    Phenol-chloroform extraction

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    How it works

    This method relies on phase separation by centrifugation ofa mix oftheaqueous sampleand a solution containing water-saturated phenol,

    chloroform and a chaotropic denaturing solution(guanidinium

    thiocyanate)resulting inanupperaqueous phaseand alowerorganic

    phase(mainly chloroform).

    Nearlyall ofthe RNA is present intheaqueous phase, while DNA and

    protein partition inthe interphaseand organic phase,respectively.

    Inalast step, RNA is recovered from theaqu

    eou

    s phase by precipitationwith 2-propanol or ethanol.

    DNA will belocated inthe interphasethus thetechnique can beused for

    DNA purificationalone.

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    Guanidinium thiocyanate denatures proteins, including RNases,and

    separates rRNA from ribosomeand dna from histones.

    Inthe presence of chloroform orBCP(bromochloropropane),these

    solvents separateentirely into two phases thatarerecognized bytheir

    color:a clear,upperaqueous phase(containingthenucleic acids)anda bright pinklowerphase(containingthe proteins dissolved in

    phenoland thelipids dissolved in chloroform).

    The majordownside is that Phenol and chloroform are both

    hazardous and inconvenient materials,and theextraction is often

    morelaborious, so inrecentyears many companies now offer

    alternative ways to isolate DNA.

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    Column-based nucleic acid purificationIt is a solid phaseextraction method to quickly purify nucleic

    acids.

    This method relies onthe factthatthe nucleic acid may bind

    (adsorption)to the solid phase(silica orother) depending onthe

    pHand the salt content ofthe buffer, which may bea Tris-EDTA

    (TE)bufferorPhosphatebuffer (used in DNA microarray

    experiments dueto thereactive amines).

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    Three stages are:

    The sample is added to the column and the nucleic acid binds

    thanks to thehigh pH and salt concentration ofthe

    binding solution, which may containbuffer,a denaturing

    agent (suchas guanidinehydrochloride)

    The column is then washed (5 mM KPO4 pH 8.0 orsimilar,

    80% EtOH)

    The column can be eluted with bufferorsimply water

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    How does this method of purification work?

    A sample(this may beanything from purified cells to atissue

    specimen collected inthe field only moments earlier) is lysed.

    Theresultant mix of proteins, DNA,phospholipids,etc., is thenrun

    throughthe channel wherethe DNA is adsorbed by silica surface inthe presence of solutions withhigh ionic strength.

    Thehighest DNA adsorptionefficiencies are shownto occurinthe

    presence of bu

    ffersolu

    tion with pHat orbelow pKa ofthe su

    rfacesilanol groups. Althoughtheexact method forthis interaction is not

    wellknown, one possibleexplanation involves reduction ofthe

    silicas surfaces negative charge dueto thehigh ionic strength ofthe

    buffer.

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    This decrease in surface chargeleads to a decrease intheelectrostaticrepulsion betweenthenegatively charged DNA and thenegatively charged

    silica. Meanwhile,the bufferalso reduces theactivity of waterby formatting

    hydrated ions.

    This leads to the silica surfaceand DNA becoming dehydrated. These

    conditions lead to anenergetically favorable situation forDNA to adsorb to

    the silica surface.

    A betterexplanation ofhow DNA binds to silica is based ontheaction of

    GuanidiumHCl(GuHCl), whichacts is a chaotrope. A chaotrope denatures

    biomolecules by disruptingthe shell ofhydrationaround them.

    This allow a positively charged ionto form a salt bridge betweenthe

    negatively charged silicaand thenegatively charged DNA backbone inhighsalt concentration.

    The DNA canthen be washed withhigh saltand EtOH,and ultimately

    eluted withlow salt.

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    QIAGEN QIAamp VIRAL RNA

    Kit contains :

    Mini spin column

    Collectiontube

    Buffers AVL

    AW1

    AW2

    AVE CarrierRNA

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    Pipette560 l of prepared BufferAVL containingcarrierRNA into a1.5 ml micro centrifugetube.

    Add 140 lbody fluid to theBufferAVL carrierRNA

    Mix by pulsevortexing for15 s.

    Incubateatroom temperature(15-25C) for10 min.Briefly centrifugethetubeto remove drops from the

    inside ofthelid.

    Role of AVL

    The sample is firstlysed underthehighly denaturingconditions provided by bufferAVL to inactivate RNases

    and to ensure isolation of intactviral RNA

    Procedure

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    Why weadd carrierRNA?y Firstly, itenhances binding ofviralnucleic acids to the

    QIAamp mini membrane,especially iftherearevery few

    target molecu

    les inthe sample.y Secondlytheaddition oflargeamounts of carrierRNA

    reduces the chance ofviral RNA degradation intherare

    eventthat Rnase molecules escape denaturation by

    chaotropic salts and detergents in bu

    fferAVL.y If carrierRNA is notadded to bufferAVL this maylead to

    reduced viral RNA recovery.

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    Add 560 l ofethanol(96-100%)to the sample,and

    mix by pu

    lse-vortexing for15 s.onlyethanol should beused since otheralcohols mayresult inreduced RNA yield and purity.

    Aftermixing, briefly centrifugethetubeto remove

    drops from thelid.

    Carefullyapply630 l ofthe solutionto the Mini SpinColumn-ina2ml Collectiontube-without wettingthe

    rim.

    Closethe cap,and centrifugeat 8000 rpm for1 min.

    Placethe spin column into a clean2ml collectiontubeand discard thetube containingthe filtrate.

    Carefully openthe Mini Spin columnand repeatthe

    step .

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    Carefully openthe Mini Spin Columnand add 500 l of

    BufferAW1.WASHBUFFER- PROTEIN DENATURING CHEMICALS

    Closethe cap and centrifugeat 8000rpm for1 min.

    Placethe Mini Spin Column ina clean2 ml collection

    tubeand discard thetube containingthe filtrate.Carefully openthe Mini Spin Columnand add 500 l of

    BufferAW2.Closethe cap and centrifugeat13,000 rpm

    for4 min.

    INCREASES THE AFFINITY BINDING OF RNA, AND CREATES

    AN ENVIRONMENT FOR RNA BINDING TO SILICA SURFACE

    Placethe Mini Spin Column into a clean,labeled 1.5ml

    micro centrifugetube.

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    Discard the old collectiontube containingthe filtrate.

    Carefully openthe Spin Column & add 60 l ofBufferAVEequilibrated to room temperature.

    It is elution buffernothing but Rnase, Dnase free water.

    Closethe cap and incubateatroom temperature for1 min.

    Centrifugeat 8000rpm for1 min.

    Stored theviral RNA at-20 to -70 degree.

    RNA loss is a majorcause ofextraction failureand commonly

    occurs duringextraction of RNA from samples using column.

    Morethan30-40% ofthe RNA is lost dueto insufficient binding

    ofthe RNA, orincompleteelution ofthe RNA from the column.Hence,anon-binding,non-elutionextraction method, which

    eliminates the RNA loss and maximizes theyield of RNA should

    be developed.

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    2

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    Quantification:Abundance in weight: spectroscopic quantification

    Absoluteabundance innumber: Q-PCR

    Size: Gelelectrophoresis

    Synthesis:

    Denovo: Oligonu

    cleotide synthesisAmplification: PCR

    Other Applications:

    Nucleic acid simulations

    DNA sequencing

    Expression cloning

    Southern blot

    northern blot

    Fluorescent in situhybridization

    several Bioinformatics methods, suchas RNA structure prediction

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    THANK YOU

    [email protected]