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