principles of dna isolation & purification
TRANSCRIPT
-
8/19/2019 Principles of Dna Isolation & Purification
1/47
PRINCIPLES OF DNA
ISOLATION & PURIFICATION
DNA can be isolated from any
ncleated cell!
-
8/19/2019 Principles of Dna Isolation & Purification
2/47
Sorces of DNA inclde
" #lood
" #ccal cells
" Cltred cells" #acterial $lasmids
" #io$sies
" Forensic sam$les i!e! body flids% airfollicles% bone & teet roots!
-
8/19/2019 Principles of Dna Isolation & Purification
3/47
Potential Sources of DNA
" #lood '(ite blood cells)
" Semen 'S$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)
-
8/19/2019 Principles of Dna Isolation & Purification
4/47
Biological Sources: All nucleated cells.
Blood
Hair Root
Saliva
Bone
Extraction
Semen
-
8/19/2019 Principles of Dna Isolation & Purification
5/47
#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!
-
8/19/2019 Principles of Dna Isolation & Purification
6/47
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
-
8/19/2019 Principles of Dna Isolation & Purification
7/47
;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!
-
8/19/2019 Principles of Dna Isolation & Purification
8/47
;ettin. rid of te $rotein
" Organic solvent extraction sin. eBal 0olme TE3sat!$enol
-
8/19/2019 Principles of Dna Isolation & Purification
9/47
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 $roteins by eatin.
-
8/19/2019 Principles of Dna Isolation & Purification
10/47
Preci$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!
-
8/19/2019 Principles of Dna Isolation & Purification
11/47
Bacterial Cells
Or tissue culture cells
Or blood
Or flies………..
HOW?
Extract
Cells
Pure DNA
Or.anic
e2traction
-
8/19/2019 Principles of Dna Isolation & Purification
12/47
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
-
8/19/2019 Principles of Dna Isolation & Purification
13/47
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
-
8/19/2019 Principles of Dna Isolation & Purification
14/47
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
-
8/19/2019 Principles of Dna Isolation & Purification
15/47
" 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
$ellet '*O%
TE% etc!)
Ste$ @
-
8/19/2019 Principles of Dna Isolation & Purification
16/47
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!.! $enolGcloroform e2traction)!
-
8/19/2019 Principles of Dna Isolation & Purification
17/47
Ncleic Acid Analysis
" DNA or RNA is caracteri7ed sin.
se0eral different metods for assessin.
Bantity% Bality% and moleclar si7e!
1 U- s$ectro$otometry
1 A.arose .el electro$oresis
1 Florometry
1 Colorimetric blottin.
-
8/19/2019 Principles of Dna Isolation & Purification
18/47
DNA &urity and Concentration
Spectrophotometry
Absorbance ma2imm
for ncleic acids ?6 nm
for $roteins H6 nm
" Concentration of DNA 1 at ?6 nm
" Prity of DNA< ratio of ?6GH6 nm
-
8/19/2019 Principles of Dna Isolation & Purification
19/47
8antity from U-
S$ectro$otometry
" DNA and RNA absorb ma2imally at ?6
nm!
" Proteins absorb at H6 nm!
" #ac,.rond scatter absorbs at 6 nm!
-
8/19/2019 Principles of Dna Isolation & Purification
20/47
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 $ea, absorbance in te ltra0iolet
ran.e at abot ?6 nm
5 A?6 O!D! nit for dsDNA J >6 K.Gml
5 A?6 O!D! nit for ssDNA J or >6 K.Gml
5 A?6 O!D! nit for RNA J @6 K.Gml
-
8/19/2019 Principles of Dna Isolation & Purification
21/47
8antity from U-
S$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
-
8/19/2019 Principles of Dna Isolation & Purification
22/47
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
-
8/19/2019 Principles of Dna Isolation & Purification
23/47
DNA $rity
" Te $rity of te DNA is reflected in te
OD?6
-
8/19/2019 Principles of Dna Isolation & Purification
24/47
A-#%A-/# measure of purity
(A-# 0 A1-#)%(A-/# 0 A1-#)
!.2 0 -.# good DNA or RNA
3!.2 too much protein or
other contaminant (4)
8ality from U- S$ectro$otometry
-
8/19/2019 Principles of Dna Isolation & Purification
25/47
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$le
-
8/19/2019 Principles of Dna Isolation & Purification
26/47
8ality from A.arose ;el
Electro$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
-
8/19/2019 Principles of Dna Isolation & Purification
27/47
=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
-
8/19/2019 Principles of Dna Isolation & Purification
28/47
=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$ared to
te len.t of standard fra.ments
-
8/19/2019 Principles of Dna Isolation & Purification
29/47
RFLP: Electrophoresis
" DNA is 0isali7ed sin. electro$oresis" Ne.ati0ely car.ed DNA mo0es tro. a .el +it a
crrent
" Smaller DNA mo0es faster tan lar.er DNA fra.ments
-
8/19/2019 Principles of Dna Isolation & Purification
30/47
-
8/19/2019 Principles of Dna Isolation & Purification
31/47
-
8/19/2019 Principles of Dna Isolation & Purification
32/47
-
8/19/2019 Principles of Dna Isolation & Purification
33/47
+,, -" 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
-
8/19/2019 Principles of Dna Isolation & Purification
34/47
DNA 8ality from
A.arose ;el Electro$oresis
" *i. moleclar +ei.t band '@H!> ,b)
" Smearin. indicates DNA de.radation 'or
too mc DNA loaded)!
-
8/19/2019 Principles of Dna Isolation & Purification
35/47
&ambda DNA
mar7er
9uman :hole ;lood DNA
&ambda DNA cut
-
8/19/2019 Principles of Dna Isolation & Purification
36/47
smmary
" Sam$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
-
8/19/2019 Principles of Dna Isolation & Purification
37/47
&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+
-
8/19/2019 Principles of Dna Isolation & Purification
38/47
DNA
Dou-le Helix
0ncoiled
Denatured
Heat
-
8/19/2019 Principles of Dna Isolation & Purification
39/47
&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 $ri5e Ste"
6
Heat
-
8/19/2019 Principles of Dna Isolation & Purification
40/47
POL/ERASE C*AIN REACTION
'PCR)+ c(cle 2 c(cles
# c(cles
7 c(cles
A!ter #,8
heating'coolingc(cles9 %e have
oodles o! DNA
6
-
8/19/2019 Principles of Dna Isolation & Purification
41/47
-
8/19/2019 Principles of Dna Isolation & Purification
42/47
-
8/19/2019 Principles of Dna Isolation & Purification
43/47
STEPS< DNA PROFILES FRO/
#IOLO;ICAL SOURCES
Extract'$uri!(
DNA
Slot -lots vs.
R13$R
Am"li!( using
$R
;isuali5e %ith
-
8/19/2019 Principles of Dna Isolation & Purification
44/47
Reslt analysis
" IF s$ecific band visuali?e at "** 5p8 then
may identi3y this Condyloma acminate tisse
+as infected by corres$ondin. ty$e *P-!
-
8/19/2019 Principles of Dna Isolation & Purification
45/47
Paternity Cases
" (os yor daddyQ
1.
2.
1. 2.
-
8/19/2019 Principles of Dna Isolation & Purification
46/47
Homicide or
Rapes:OJ Simpson
-
8/19/2019 Principles of Dna Isolation & Purification
47/47
RFLP: Autoradiograph