principles of dna isolation & purification

8/19/2019 Principles of Dna Isolation & Purification

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PRINCIPLES OF DNA

ISOLATION & PURIFICATION

DNA can be isolated from any

ncleated cell!


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Sorces of DNA inclde

" #lood

" #ccal cells

" Cltred cells" #acterial $lasmids

" #io$sies

" Forensic sam$les i!e! body flids% airfollicles% bone & teet roots!


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Potential Sources of DNA

" #lood '(ite blood cells)

" Semen 'S$erm cells)

"*air +it roots '*air follicle cells)" S,in% dandrff 'S,in cells)

" -a.inal flids'/cosal srfaces)

" Nasal secretions '/cosal srfaces)" Urine '/cosal srfaces)

" Feces 'Di.esti0e system cells)


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Biological Sources: All nucleated cells.

Blood

Hair Root

Saliva

Bone

Extraction

Semen


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#asic rles

" Blood 1 first lyse 'e2$lode) te red blood cells +it a.entle deter.ent sc as Triton343566!

" Wash cells 1 aemo.lobin 'and oter $i.ments) inibits

restriction en7ymes and TA8 $olymerase!" (or, on ice to slo+ do+n en7ymatic $rocesses!

" (ear gloves to $rotect yor sam$les from yo99

" Autoclave all soltions and store in frid.e 'e2ce$t SDS

and or.anic sol0ents9)" :ee$ all $ellets & s$ernatants ntil yo a0e te DNA

yo +ant!


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DNA Isolation #asic Ste$s

" Cell lysis

" Remo0al of $roteins3 $rotease

3 adsor$tion or e2traction

" DNA $reci$itation by etanol" DNA diltion in +ater or bffer


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;ettin. to te DNA

" Cells 1 lyse all cells in $resence of <

" NaCl so tat DNA is stabilised and remains as a doble eli2%

" EDTA +ic celates /.== and is a co3factor of DNAse +icce+s $ DNA ra$idly%

" anionic detergent SDS +ic disr$ts te li$id layers% el$s todissol0e membranes & binds $ositi0e car.es of cromosomal

$roteins 'histones) to release te DNA into te soltion!

" Inclde a protease (proteinase K ) to di.est te $roteins

" incbate te soltion at an elevated temperature '>?oC toinibit de.radation by DNAses) for @3@ rs!


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;ettin. rid of te $rotein

" Organic solvent extraction sin. eBal 0olme TE3sat!$enol


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DNA Isolation For #asic /etods

5! Penol3cloroform e2traction

'different solbility conditions in sol0ents)

! Saltin. ot metod'$rotein $reci$itation by NaCl)

! Adsor$tion metod

'silica3.el membrane)

@! Denatration of $roteins by eatin.


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Preci$itatin. te DNA

" add !> 3 0olmes icecold !"#

ethanol$acetate to te DNA & lea0e at 36oC

o0erni.t!

" Centrif.e sam$le at 56:% 56% @6C!" Wash DNA $ellet to remo0e e2cess salt in 6

EtO* and air3dry!

" %esuspend in sterile distilled +ater 'sd+) or TE%$*!@!

" Store at @oC or fro7en at 36oC lon. term!


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Bacterial Cells

Or tissue culture cells

Or blood

Or flies………..

HOW?

Extract

Cells

Pure DNA

Or.anic

e2traction


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High MW Genomic DNA solation

Detail o! ste" #

$henol Extraction• mix sam"le %ith e&ual volume

o! sat. "henol soln

• retain a&ueous "hase

• o"tional chloro!orm'isoam(lalcohol extraction)s*

← aqueous phase

(nucleic acids)

← phenol phase

(proteins)

Typical &rocedure

5 'arvest cells

( Cell )ysis

1 *+"# SDS , proteinase -

.""o several hours/

0 &henol Extraction

1 gentle roc1ing several

hours

2 Ethanol &recipitation

" %NAse 3ollo4ed 5yproteinase -

6 %epeat &henol Extraction

and EtO' ppt


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Crde lysate containin.

ncleic acids and otercell constitents

/i2 toro.ly +it

an eBal 0olme of

or.anic sol0ent

e.g. $enol%

cloroform% or

$enol


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High MW Genomic DNA solation

Typical &rocedure

5 'arvest cells

( Cell )ysis

1 *+"# SDS , proteinase -

.""o several hours/

0 &henol Extraction

1 gentle roc1ing several

hours

2 Ethanol &recipitation

" %NAse 3ollo4ed 5yproteinase -

6 %epeat &henol Extraction

and EtO' ppt

Detail o3 step 2

EtO' &recipitation" ((+" volumes EtO'8 (*o

" high salt8 p' ""+"" centri3uge or 9spool: out


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" Pellet do+n ncleic acids!" (as $ellet +it 6 etanol to remo0e

residal salts and oter contaminants!

" Discard etanol and allo+ $ellet to dry!

After

Add alcool and salt to

$reci$itate ncleic acids

from te aBeos fraction

S$ernatant

Pellet

6 EtO*

Dissol0e

$ellet '*O%

TE% etc!)

Ste$ @


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Detail o3 step "

;sing Nucleases to %emove ;n4anted DNA or %NA

Add DNase

Add RNase

= DNase '$rotein)

= RNase '$rotein)

De$endin. on +en nclease treatment is $erformed% it may be necessary to

re$eat $rification ste$s for $rotein remo0al 'e!.! $enolGcloroform e2traction)!


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Ncleic Acid Analysis

" DNA or RNA is caracteri7ed sin.

se0eral different metods for assessin.

Bantity% Bality% and moleclar si7e!

1 U- s$ectro$otometry

1 A.arose .el electro$oresis

1 Florometry

1 Colorimetric blottin.


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DNA &urity and Concentration

Spectrophotometry

Absorbance ma2imm

for ncleic acids ?6 nm

for $roteins H6 nm

" Concentration of DNA 1 at ?6 nm

" Prity of DNA< ratio of ?6GH6 nm


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8antity from U-

S$ectro$otometry

" DNA and RNA absorb ma2imally at ?6

nm!

" Proteins absorb at H6 nm!

" #ac,.rond scatter absorbs at 6 nm!


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8antifyin. te DNA

" Te absorbance 'O!D!) of a cemical is te<" $rodct of its 'concentration) 2 'o$tical $at len.t) 2

'e2tinction coefficient% E)!" Ncleic acids a0e a $ea, absorbance in te ltra0iolet

ran.e at abot ?6 nm

5 A?6 O!D! nit for dsDNA J >6 K.Gml

5 A?6 O!D! nit for ssDNA J or >6 K.Gml

5 A?6 O!D! nit for RNA J @6 K.Gml


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8antity from U-

S$ectro$otometry

" DNAM J

'A?6 1 A6) 4 diltion factor 4 >6 K.G/l

" RNAM J

'A?6 1 A6) 4 diltion factor 4 @6 K.GmL

" Concentration J K. of DNA or RNA $er mLof ydratin. soltion


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Multiply the concentration of the

DNA or RNA sample by the

volume of hydrating solution added.

Exam"le !or DNA !"# $g%m& ' #.! m& !" $g

oncentration from *+

,pec. ($g DNA per ml

of hydrating solution)

+olume of

hydration

solution

DNA yield

8antity from U- S$ectro$otometry

Calclatin. ield


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DNA $rity

" Te $rity of te DNA is reflected in te

OD?6


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A-#%A-/# measure of purity

(A-# 0 A1-#)%(A-/# 0 A1-#)

!.2 0 -.# good DNA or RNA

3!.2 too much protein or

other contaminant (4)

8ality from U- S$ectro$otometry


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DNA &urity and Concentration

=el Electrophoresis

" DNA is stained by intercalatin. dyes in .el

'Florescent dye 3 etidim bromide)

" ;el is loadin. +it DNA standard

'its concentration is $re3e0alated)

" Com$arison of t+o li.t intensities 1

standard and or sam$le


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8ality from A.arose ;el

Electro$oresis

" ;enomic DNA<

1 6!? to 5 .el% 6!5> K.GmL etidim bromide

in .el andGor in rnnin. bffer

1 Electro$orese at 61H6 0olts% @>16 mintes!

" Total RNA<

1 5 to .el% 6!5> K.Gml etidim bromide in

.el andGor in rnnin. bffer 1 Electro$orese at H61566 0olts% 61@6 mintes!

=el Electrophoresis


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=el ElectrophoresisSe$aration metod

;el 1

sie0e strctre of $olymer molecles +it $ores

;el 3 a.arose3 $olyacrylamid

Etidim bromide is an intercalatin. dye%

binds to DNAIt creates com$le2 e2citin. $otons

after U-3e2$osre

=el Electrophoresis


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=el ElectrophoresisSe$aration metod

&%INCI&)E to =

'te ncleic acids consist of ne.ati0ely car.ed $os$ate .ro$s)

" DNA3rate in .el de$ends on DNA3fra.ment len.t

in indirect $ro$ortionTe len.t of n,no+n fra.ments is com$ared to

te len.t of standard fra.ments


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RFLP: Electrophoresis

" DNA is 0isali7ed sin. electro$oresis" Ne.ati0ely car.ed DNA mo0es tro. a .el +it a

crrent

" Smaller DNA mo0es faster tan lar.er DNA fra.ments


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+,, -" ladder

+ - ladder/am-da DNA cut

%ith Hind /am-da DNA

5/6"## bp

(5/." 7b)

!-6-!/ bp

-16!1# bp

865! bp

6""2 bp

561! bp

-61-- bp

-6#-2 bp"!2 bp

!61 bp

16#"5 bp

6#!/ bp

!## bp

1## bp

## bp

!6### bp

!6"## bp

!6#!/ bp

-6#1 bp

DNA Si?e 3rom Agarose =el Electrophoresis<

Compares un1no4n DNA to 1no4n si?e

standards


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DNA 8ality from

A.arose ;el Electro$oresis

" *i. moleclar +ei.t band '@H!> ,b)

" Smearin. indicates DNA de.radation 'or

too mc DNA loaded)!


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&ambda DNA

mar7er

9uman :hole ;lood DNA

&ambda DNA cut


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smmary

" Sam$le for DNA e2traction

" Lysis of cells at ele0ated tem$eratre =

deter.ent = en7yme in salt bffer

" Remo0al of celllar $roteins

" Preci$itation of ncleic acids +it etanol

" 8antitation and $rity measrement ofDNA


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&O)@E%ASE C'AIN

%EACTION .&C%/9molecular eroxing:

" The process 5y 4hich scientists ma1emillions o3 copies o3 speci3ic pieces o3

DNA+

" The si?e o3 these eroxed DNA3ragments di33er 3rom one person to

another+


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DNA

Dou-le Helix

0ncoiled

Denatured

Heat


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&O)@E%ASE C'AIN %EACTION

.&C%/

1em"late DNA

Denature

$ol(merase

Extension

2 co"ies o! original tem"late3DNA has dou-led4

Anneal

$rimers

Heat

No-el $ri5e Ste"

6

Heat


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POL/ERASE C*AIN REACTION

'PCR)+ c(cle 2 c(cles

# c(cles

7 c(cles

A!ter #,8

heating'coolingc(cles9 %e have

oodles o! DNA

6


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STEPS< DNA PROFILES FRO/

#IOLO;ICAL SOURCES

Extract'$uri!(

DNA

Slot -lots vs.

R13$R

Am"li!( using

$R

;isuali5e %ith


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Reslt analysis

" IF s$ecific band visuali?e at "** 5p8 then

may identi3y this Condyloma acminate tisse

+as infected by corres$ondin. ty$e *P-!


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Paternity Cases

" (os yor daddyQ

1.

2.

1. 2.


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Homicide or

Rapes:OJ Simpson


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RFLP: Autoradiograph

principles of dna isolation & purification

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