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ASPECTOS GENERALES DE AISLAMIENTO Y PURIFICACIN DE ADN

1.1 Introduccin
El aislai!nto d! ADN !s una t"cnica !s!ncial !n la #iolo$%a ol!cular& El aislai!nto
d! ADN d! alto '!so ol!cular s! (a )u!lto u* i'ortant! con la cr!ci!nt! d!anda
d! la (u!lla dactilar $!n"tica+ 'olior,iso d! lon$itud d! ,ra$!ntos d! r!striccin
-RFLP.+ la construccin d! #i#liot!cas $!nicas o d! s!cu!nciacin * an/lisis d!
PCR !n los la#oratorios d! in)!sti$acin * la industria& Ad!/s+ !l aislai!nto d! ADN
!s !l 'ri!r 'aso !n !l !studio d! s!cu!ncias d! ADN !s'!c%,icas d!ntro d! una
'o#lacin d! ADN co'l!0o+ * !n !l an/lisis d! la !structura d!l $!noa * la !1'r!sin
$"nica& La cantidad+ calidad * la int!$ridad d!l ADN a,!ctar/ dir!cta!nt! !stos
r!sultados &DNA constitu*! un '!2u!3o 'orc!nta0! d!l at!rial c!lular * s! locali4a
usual!nt! !n una 'art! d!,inida d! las c"lulas 'rocariotas+ !l ADN s! locali4a !n !l
nucl!tido 2u! no !st/ s!'arado d!l r!sto d! la sa)ia d! la c"lula 'or una !#rana&
En las c"lulas !ucariotas+ la a*or 'art! d! ADN s! locali4a !n !l n5cl!o+ un or$/nulo
2u! !st/ s!'arado d!l cito'lasa 'or una !#rana& El n5cl!o conti!n!
a'ro1iada!nt! 678 d!l ADN c!lular total+ !l ADN r!stant! !st/ !n otros or$/n!los
tal!s coo itocondrias+ cloro'lastos o cin!tocoros& En )irus * #act!ri,a$os+ !l ADN
!st/ !nca'sulado 'or una ca'a d! 'rot!%na+ * constitu*! !ntr! !l 97 * !l :7 'or ci!nto
d! la asa total d!l )irin& En las c"lulas 'rocariotas * !ucariotas ADN constitu*! slo
!l ;8 d! la asa total d! la c"lula& La co'osicin a'ro1iada d! c"lulas d! E& coli *
c"lulas (uanas -<!La. 2u! s! di)id!n r/'ida!nt! s! 'r!s!nta !n la Ta#la I&
El 'ro'sito d! aislai!nto d! ADN !s s!'arar !l ADN d! todos los co'on!nt!s d! la
c"lula listados !n la Ta#la ; 2u! r!sulta !n una 'r!'aracin d! ADN (oo$"n!a 2u!
r!'r!s!nta la totalidad d! la in,oracin $!n"tica cont!nida d!ntro d! una c"lula& No
(a* nin$una di,icultad !n la s!'aracin d! ADN d! ol"culas '!2u!3as *a 2u! !l '!so
ol!cular d!l ADN !s u* $rand!& En cons!cu!ncia+ los 'rinci'al!s co'on!nt!s
c!lular!s 2u! ti!n!n 2u! s!r r!tirado durant! la 'uri,icacin d! ADN son las 'rot!%nas *
ARN&
R!2uisitos $!n!ral!s d! aislai!nto d! ADN !,ica4 son=
> El "todo d!#! 'roducir ADN sin a*or!s containant!s+ 'rot!%nas
* !l ARN&
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G!n!ral As'!cts o, DNA Isolation and Puri,ication
STEFAN SURZYCKI ~
;&;? Introduction
DNA isolation is an essential technique in molecular biology. Isolation of
high-molecular weight DNA has become very important with the increasing
demand for DNA fingerprinting, restriction fragment length polymorphism
(!"#$, construction of genomic or sequencing libraries and #% analysis in
research laboratories and industry. Also, DNA isolation is the first step in the
study of specific DNA sequences within a comple& DNA population, and in
the analysis of genome structure and gene e&pression. 'he quantity, quality
and integrityof DNA will directly affect these results.DNA constitutes a small
percentage of the cell material and is usua(y locali)ed in a defined part of the
cell.In pro*aryotic cells, DNA islocali)ed in the nucleoid thatis not separated
from the rest of the ce( sap by a membrane. In eu*aryotic cells, the bul* of
DNA is locali)edin the nucleus, an organe(e that is separated from the cy-
toplasm by a membrane. 'he nucleus contains about +,- of the total ce(ular
DNA, the remaining DNA is in other organelles li*e mitochondria, chloro-
plasts or *inetochores. In viruses and bacteriophages, the DNA is encapsu-
lated by a protein coat, and constitutes between ., and /, percent of the total
mass of the virion. In pro*aryotic and eu*aryotic ce(s DNA constitutes only
about I - of the total mass of the cell. 'he,appro&imate composition of ra-
pidly dividing 0. coli cells and human cells ((e"a$ is presented in 'able I.
'he purpose of DNA isolation is to separate DNA from all the compo-
nents of the ce( listed in 'able I resulting in a homogeneous DNA prepara-
tion that represents the entire genetic information contained within a cell.
'here is no difficulty in separating DNA from sma( molecules since the
molecular weight of DNA is very large. %onsequently, the main ce(ular
components that have to be removed during DNA purification are protein
and NA. 1eneral requirem2nts of effective DNA isolation are3
o 'he method should yield DNA without ma4or contaminants, protein
and NA.
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