1isolasi Dna

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O G O

DNA ISOLATION

Riandini Aisyah2010


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Relevance of DNA isolation

Isolation of DNA is often the first step before further analysis

DNA profiling (forensics)

Cloning

Disease diagnosisDNA sequencing

enetically !odified organis!s ("#) $ agriculture%

&har!aceutical

'niron!ental testing% bioterroris!


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Structure of the cell

tructure of the cell

&las!a !e!brane and !e!branes of organelles

nuclear

'nelope included

DNA located in nucleus

A lot of proteins around

"itochondrial DNA


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Extraction of genomic DNA

Cell collection Add *ysis buffer to cells to brea+ open cell

and nuclear !e!branes and release

nuclear contents

Digest sa!ple ,ith protease to degrade

proteins

&recipitate DNA ,ith cold alcohol in highsalt


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Lysis buffer

*ysis buffer -

.0 !" /ris$CI% p 0 to !aintain the p

of the solution at a leel ,here DNA is stable

13 D (sodiu! dedo4yl sulfad) to brea+ open the cell

and nuclear

!e!branes% allo,ing the DNA to be released

into the solution (D also denatures and unfolds

proteins% !a+ing the! !ore susceptible to protease

cleaage)


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Why a !rotease"

&rotease destroys nuclear proteins thatbind DNA and cytoplas!ic en5y!es that

brea+do,n and destroy DNA

&rotease treat!ent increases the a!ount

of intact DNA that is e4tracted


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Aing salt

/he addition of NaCI allo,s the DNA !olecules to co!e

together instead of repelling each other% thus !a+ing iteasier for DNA to precipitate out of solution ,hen alcohol

is added

Na6 ions bind to the phosphate groups of DNA

!olecules% neutrali5ing the electric charge of the DNA!olecules

#ur protease solution already contains salt


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#reci!itation of DNA

DNA does not dissole (larut) in alcohol

Addition of cold alcohol !a+es the DNA clu!p together

and precipitate out of solution

&recipitated DNA !olecules appear as long pieces of

fluffy (soft%light)% stringy (sprt +a,at)% ,eb$li+e strands

"icroscopic o4ygen bubbles 7aggregate8 together% as the

DNA precipitates

/he larger% isible air bubbles 7lift8 the DNA out of

solution% fro! the aqueous into the organic phase

/he DNA in the glass ial can last for years


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'4traction of DNA basically consists of four

!a9or steps -

: &reparation of a cell e4tract

: &urification of DNA fro! cell e4tract

: Concentration of DNA sa!ples

: "easure!ent of purity and DNA

concentration

ind,iani;yahooco!


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$% #re!aration of a cell extract&

/o e4tract DNA fro! a tissue


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'% #urification of DNA from cell extr act

In addition to DNA the cell e4tract ,ill contain significantquantities of protein and RNA

A ariety of procedures can be used to re!oe theseconta!inants% leaing the DNA in a pure for!

/he standard ,ay to de$proteini5e a cell e4tract is to add

phenol or a 1-1 !i4ture of phenol-chlorofor! /hese organic solents precipitate proteins but leae the

nucleic acids in aqueous solutions /he aqueous solution of nucleic acid can be re!oed

,ith a pipette

/he effectie ,ay to re!oe RNA is ,ith the en5y!eribonuclease% ,hich ,ill rapidly degrade these !oleculesinto ribonucleotide subunits


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Isolation of DNA

ind,iani;yahooco!

http://www.agen.ufl.edu/~chyn/age4660/lect/lect_07/FG06_054.GIFhttp://www.agen.ufl.edu/~chyn/age4660/lect/lect_07/FG06_054.GIF


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(% )oncentration of DNA sam!les

/he !ost frequently used !ethod ofconcentration is ethanol precipitation

In the presence of salt and at a te!perature of$20 >C or less% absolute ethanol ,ill efficientlyprecipitate poly!eric nucleic acids

?ith a concentrated solution of DNA one canuse a glass rod to pull out the adhering DNA

strands ,hile for dilute solutions precipitatedDNA can be collected by centrifugation andredissoling in an appropriate olu!e of ,ater

ind,iani;yahooco!


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*el Electro!horesis A!!aratus

ind,iani;yahooco!

http://www.agen.ufl.edu/~chyn/age4660/lect/lect_07/PH06_001.GIFhttp://www.agen.ufl.edu/~chyn/age4660/lect/lect_07/PH06_001.GIF


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*el Results

ind,iani;yahooco!


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-. Absor!tion S!ectrum of DNA

ind,iani;yahooco!


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DNA Labeling /ith Dyes

ind,iani;yahooco!


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)esium *raient )entrifugation

ind,iani;yahooco!


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DNA ,elting

ind,iani;yahooco!


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0eating anc )ooling of DNA 1Thermocycler2

ind,iani;yahooco!


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DNA 0ybrii3ation

ind,iani;yahooco!


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#lot 4rom DNA Analy3er

ind,iani;yahooco!


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ind,iani;yahooco!


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Recognition sequences of a fe, restriction endonucleases

Organism En3yme esignation Recognitionse5uence

Escherichia coli 'coRI A ↓ AA//C

Escherichia coli 'coRII $C ↓ CCA

Haemophilus

influenzae

indII &$C /&&y↓ &uAC

Haemophilushemolyticus

haI CC C↓ C

Bacillus subtilus EsuRI C ↓ CC

Brevibacterium

albidumEaII C /↓ CCA

Thermus aquaticus /aql /A /↓ CA

Arrows indicate the sites of enzymatic attack. Underlines indicate the site of

methylation modification!. " # $uanine% & # cytosine% A # adenine T #

thymine% 'u # any purine% 'y # any pyrimidine. (nly the )* → +, sequence is

shown. ind,iani;yahooco!


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ind,iani;yahooco!


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Isolasi DNA ba6teri

e9u!lah +ecil dari total DNA% ter!asu+ /i plas!id dan

DNA +ro!oso! dari A$robacterium disiap+an dengan!etode Fado dan *iu (G1)

el$sel ditu!buh+an se!ala!an dala! *$Eroth pada

suhu 2> C hingga densitas opti+ 0 pada B00 n! dandi9adi+an pelet !elalui sentrifugasi pada 2. + 4 gsela!a H !enit

&elet sel disuspensi+an dala! 1 !l larutan penyangga '(G0 n" /ris$asetat% p H% !engandung 2 !" 'D/A)


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el$sel tersebut dihancur+an dengan cara

!ena!bah+an 2 !l larutan penghancur (J3 D dala!.0 !" /ris$Na#% p 12B)% dan dica!pur denganpengadu+an sing+at *arutan tersebut dipanas+an padasuhu B.> C sela!a 10 !enit dala! water bath dandita!bah+an dua olu!e larutan fenol- +lorofor! (1-1% C)

Kase cair bagian atas dialir+an +e dala! tabung barudan DNA di9adi+an pelet dengan !ena!bah+an 01

olu!e natriu! asetat J " (p G) dan 2. olu!eetanolDNA dala! bentu+ pelet disuspensi +e!bali dala! 100

!l larutan penyangga /' (/ris$Cl 10 n"% p 0%!engandung 'D/A 1 !")


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&las!id lainnya% seperti p@C1% diisolasi denganprosedur lisis al+alin D (B)

el ba+teri di+ultur se!ala!an dala! !edia *E atau /Eyang !engandung antibioti+ yang sesuai Fultur tersebut(1. !l) disentrifugasi dan peletnya disuspensi dala!100 l larutan penyangga (/ris$Cl .0!"% p 0%!engandung glu+osa .0 !" dan 'D/A 10 !")

etelah disi!pan dala! es sela!a . !enit% suspensitersebut dita!bah+an dengan 200l larutan penghancur(Na# 0 "% /riton L$100 G3)% dica!pur dengan caradibali+ beberapa +ali dan dite!pat+an dala! es sela!a. !enit Fe!udian% dita!bah+an 1.0 l natriu! asetat J" (p G) dan dite!pat+an dala! es sela!a . !enit


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Ca!puran itu disentrifugasi pada suhu ruangan sela!a. !enit upernatannya dituang+an dala! tabungmicrocentrifu$e bersih% dan dita!bah+an 1 olu!e (G.0l) isopropanol% dica!pur dengan cara !e!bali+ tabungbeberapa +ali% dii+uti dengan in+ubasi dala! suhuruangan sela!a .$10 !enit

*arutan tersebut disentrifugasi pada 1J + 4 g sela!a 10!enit untu+ !engubah plas!id DNA !en9adi peletetelah pengeringan% plas!id DNA disuspensi +e!balidala! .0 l larutan /' Mi+a perlu% RNA yang!eng+onta!inasi pada endapan dihilang+an dengan

prosedur beri+ut DNA plas!id dilarut+an dala! 00 llarutan /'% +e!udian dita!bah+an 100 l Rnase A(dipanas+an% 100 g


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*arutan tersebut diin+ubasi pada suhu JH> C sela!a J0!enit% dan +e!udian disentrifugasi untu+ !enghilang+anbahan yang tida+ dapat larut

*arutan polietilen gli+ol B000 (203 dala! NaCl 2. "%B00 l) dita!bah+an +e dala! supernatan etelahin+ubasi dala! es sela!a dua 9a!% larutan tersebutdisentrifugasi pada 1J + 4 g sela!a . !enit

'ndapannya dilarut+an dala! .00 l larutan /' dandie+stra+ dengan fenol% fenol-+lorofor! (1-1%


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el$sel ba+teri di+ultur hingga 9enuh dala! 100 !l !ediu!*E el$sel tersebut di9adi+an pelet dengan sentrifugasi J. +4 g sela!a 10 !enit% dan +e!udian disuspensi +e!bali dala!. !l larutan penyangga /'

uspensi ba+teri dita!bah+an dengan 0. !l D 103 dan

.0 l proteinase F (20!g C NaCl (. "% 1% !l) danlarutan C/AE C sela!a 20 !enit

Ca!puran itu dita!bah+an dengan +lorofor!-isoa!ilal+oholdengan olu!e yang sa!a (2G-1%


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DNA di9adi+an pelet dengan pena!bahan 0B olu!e isopropanol

dan disentrifugasi pada + 4 g sela!a .$10 !enit

&elet DNA dicuci dengan etanol H03% di+ering+an dan disuspensi+e!bali dala! G !l larutan penyangga /'% dii+uti denganpena!bahan GJ g CsCl dan 200 l ethidiu! bro!ida (10!g

Ca!puran itu +e!udian dialir+an +e tabung centrifu$e yang dapatditutup rapat (sealable centrifu$e tube) dan diputar dala! rotorerti+al sela!a 1 9a! pada B00 + 4 g pada suhu 1.> C

Diba,ah sinar @ pita DNA +ro!oso!al yang ada dala! tabung

centrifu$e dipindah+an dengan !engguna+an alat sunti+ dandie+stra+si dengan ca!puran /'-isoa!ilal+ohol (1-1%


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Ele6troforesis *el Agarose

'le+troforesis gel Agarose dila+u+an !enurut !etode

yang telah dilapor+an(1.) Eai+ gel agarose 03ataupun 13 dapat diguna+an% tergantung pada u+uranDNA yang a+an dipisah+an

Diguna+an siste! larutan penyangga dengan +adargara! rendah terbuat dari /ris$asetat G0 !" (p H)

dan sodiu! 'D/A (larutan penyangga /A') 2!" e!ua proses ele+troforesis diperlihat+an pada sebuahaparatus hori5ontal dan dila+u+an dengan daya 100 sela!a 1$J 9a!

etelah ele+troforesis dan pe,arnaan dala! larutan

ethidiu! bro!ida (0. g


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