Nucleic Acids Resea ch, 2009, 1–6
doi:10.1093/na /gkp539
Sepa a ion o 1–23-kb complemen a y DNA
s ands by u ea–aga ose gel elec opho esis
E
´ a Hegedu¨s
1
, End e Ko
´kai
2
, Alexande Ko lya
3
, Vik o Domb a
´di
2,4
and Ga
´bo Szabo
´
1,
*
1
Depa men o Biophysics and Cell Biology,
2
Depa men o Medical Chemis y, Uni e si y o Deb ecen, 4012
Deb ecen, Nagye dei k . 98, Hunga y,
3
Depa men o Biochemis y, Geo ge S. Wise Facul y o Li e Sciences
and Nano echnology Cen e , Tel A i Uni e si y, Rama A i 69978, Is ael and
4
HAS Cell Biology and
Signalling Resea ch G oup, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, 4012 Deb ecen,
Nagye dei k . 98, Hunga y
Recei ed No embe 26, 2008; Re ised June 8, 2009; Accep ed June 9, 2009
ABSTRACT
Double-s anded (ds), as well as dena u ed, single-
s anded (ss) DNA samples can be analyzed on
u ea–aga ose gels. He e we epo ha a e dena-
u a ion by hea in he p esence o 8 M u ea, he wo
s ands o he same ds DNA agmen o ~1–20-kb
size mig a e di e en ly in 1 M u ea con aining aga-
ose gels. The wo s ands a e eadily dis inguished
on Sou he n blo s by ss-speci ic p obes. The di e -
en mig a ion o he wo s ands could be a ibu ed
o hei di e en , base composi ion-dependen
con o ma ion impinging on he elec opho e ic
mobili y o he ss molecules. This phenomenon
can be exploi ed o he e icien p epa a ion o
s and-speci ic p obes and o he sepa a ion o
he complemen a y DNA s ands o subsequen
analysis, o e ing a new ool o a ious cell biolog-
ical esea ch a eas.
INTRODUCTION
Gel elec opho esis is a powe ul, ye , con enien ool
ou inely used o sepa a e nucleic acids on he basis o
diffe ences in hei size, as well as local and global con-
o ma ional cha ac e is ics. Fo molecules smalle han
he po e size o he gel, he elec opho e ic mobili y is
adequa ely desc ibed by he Ogs on sie ing mechanism
(1). Longe molecules ha exceed he olume o a single
po e snake h ough he gel ma ices in an end-on ashion
(2–4), while he DNA molecules end o s ay o ien ed
pa allel o he elec ic field (5). The model o his la -
e elec opho e ic p ocess, called ep a ion, is based
on he concep o a ube h ough which he nucleic
acid chain passes ia andom s e ching and sh inking
[(6–8) and e e ences he ein]. In 1% aga ose gels, he
double-s anded (ds) DNA molecules appea o ollow
he Ogs on app oxima ion below 4-kb size, while hey
a e expec ed o beha e as p edic ed by he ep a ion model
a la ge sizes (6,7). In addi ion o size and con o ma ional
cha ac e is ics, he handedness o supe coiling influences
elec opho e ic mobili y (9), a phenomenon lacking molec-
ula explana ion. The ela i ely uns able single-s anded
(ss) nucleic acid molecules appea o o m coiled s uc-
u es wi h size pa ame e s ha a e sensi i e no only o
base composi ion, bu also sequence in a size ange o
up o 2–300-bp leng h; his phenomenon is u ilized
in single-s and con o ma ion analysis pe o med usu-
ally in polyac ylamide gels (10–12). O ien a ion o
he gel ma ix i sel in he elec ic field has also been
ecognized as a ac o influencing elec opho e ic mobil-
i y (5). The a e age po e size is ypically 200–500 nm
o aga ose, and i exceeds ha o ac ylamide gels ha
anges om 5 o 100 nm, depending on he condi ions
and me hods o assessmen used (13). Polyac ylamide
appea s o be chemically ine , while he hyd oxyl
g oups o aga ose may pa icipa e in ansien H-bonding
du ing mig a ion.
The elec opho e ic sepa a ion o u ea/hea -dena u ed
and non-dena u ed (conside ed ss and ds, espec i ely)
nucleic acids in he same 1 M u ea-con aining aga ose
gels was fi s desc ibed by Ma e na e al. (14): hey
obse ed a diffe ence in he mig a ion o ds s. ss mole-
cules o he same size and band duplica ion a e dena-
u a ion in he case o one o he PCR agmen s analyzed,
wi hou commen ing on he s andedness dependence o
elec opho e ic mig a ion documen ed and cha ac e ized
in de ail he ein. The gene al belie s ill conside s u ea as
a dena u an unsui able o use in aga ose gels (15). He e
we demons a e ha his sepa a ion sys em can be e y
use ul in applica ions equi ing he sepa a ion o he com-
plemen a y DNA s ands in an unexpec edly b oad size-
ange, opening new a eas o applica ion.
*To whom co espondence should be add essed. Tel: +36 52 455 866; Fax: +36 52 532 201; Email: [email p o ec ed]; [email p o ec ed]
ß2009 The Au ho (s)
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion Non-Comme cial License (h p://c ea i ecommons.o g/licenses/
by-nc/2.0/uk/) which pe mi s un es ic ed non-comme cial use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
Nucleic Acids Resea ch Ad ance Access published June 23, 2009
MATERIALS AND METHODS
Aga ose-embedded yeas genomic DNA
The Saccha omyces ce e isiae WDHY 199 (MATa,
leu2-3,112 p1-289 u a3-52 his7-2 lys1-1) cells we e
g own and he p epa a ion o aga ose-plugs con aining
he yeas sphe oplas s was ca ied ou as p e iously
desc ibed (16). Fo es ic ion enzyme diges ion, he
plugs we e p eincuba ed in he app op ia e 1 es ic ion
enzyme buffe s h ee imes o 1 h each, hen incuba ed
wi h 150 U/ml SmaI o Nb.Bpu10I (Fe men as Li e
Science, Ma yland, USA) in 200 ml o he same buffe a
378C, o 1.5 h. Fo S1 nuclease ea men , 1S1 buffe
was used o washing o he plugs be o e diges ion by
500 U/ml o he enzyme (P omega Li e Science,
Madison, USA) a 378C, o 1.5 h. The plugs we e finally
equilib a ed wi h TE buffe be o e elec opho esis.
PCR ampli ica ion o S. ce e isae DNA segmen s
PCR was pe o med using 1.25 U o he Long PCR
Enzyme mix (Fe men as) in 50 mlo 1buffe supplemen-
ed wi h 1.5 mM MgCl
2
, con aining, 20 pmol o each
p ime (In eg a ed DNA Technologies, Co al ille, IA,
USA), he dNTPs (P omega) a 0.25 mM concen a ion
and 300 ng S. ce e isae genomic DNA p epa ed as des-
c ibed ea lie (16). Each o wa d p ime (see Tables 1–2
o Supplemen a y da a) was used in pai wi h he e e se
p1R p ime esul ing in a iable leng h o amplicons,
o e lapping a hei 30-ends defined by he common
e e se p ime .
Sample p epa a ion o u ea–aga ose gel elec opho esis
Be o e loading he DNA samples on he gels, ei he 5 ml
DNA (0.1–1 mg) solu ion was added o 25 ml u ea–LB
[0.5 mg/ml b omephenol blue (Sigma), 8 M u ea (Sigma),
1% ( / ) NP-40 (Calbiochem), 1mM T is pH 8] o when
DNA was embedded in o aga ose plugs, he blocks we e
soaked in o eshly p epa ed 8 M u ea solu ion/TE a
oom empe a u e o 45 min. These samples we e ei he
loaded wi hou dena u a ion, o we e hea -dena u ed a
808C o 5 min and hen loaded on he same gel.
S anda d and u ea/hea -dena u ing aga ose gel
elec opho esis
Fo s anda d, non-dena u ing gel elec opho esis, 1.2%
aga ose gels (SeaKem) we e p epa ed in 1TAE
(40 mM T is-ace a e, 1 mM EDTA, pH 8); he elec o-
pho esis buffe was also 1TAE. Fo u ea/hea -
dena u ing gel elec opho esis, 1.2% aga ose gels con ain-
ing 1 M u ea we e p epa ed; he elec opho esis buffe was
1TAE supplemen ed wi h 1 M u ea. Elec opho esis was
ca ied ou in he cold oom, a 48C and 55 V, o 12 h.
A e gel elec opho esis aga ose gels we e s ained wi h
0.5 mg/ml e hidium b omide (Eb ; P omega) o 30 min,
bu he u ea–aga ose gels we e washed in 1TAE o
emo e u ea, hen soaked in 100 mM NaCl solu ion and
s ained wi h 0.5 mg/ml EB o 30 min. In eassocia ion
expe imen s, aga ose blocks con aining ss and ds ag-
men s we e excised, hea ea ed a 958C o 5 min, hen
allowed o ena u e a 458C o 30 min be o e loading
hem on s anda d aga ose gels. Molecula mass ma ke s
we e lambda HindIII agmen s and he 1 kb ladde
o Fe men as.
Sou he n blo wi h DNA-speci ic p obes
The s anda d and u ea/hea -dena u ing aga ose gels we e
ans e ed o Hybond-N
+
nylon memb anes (Ame sham
Pha macia Bio ech) using a BIO-RAD acuum blo e .
The memb anes we e d ied o 30 min a 808C and UV
c oss-linked (1.2 10
5
mJ/cm
2
). The blo ed, dena u ed
DNA was p ehyb idized o 3 hou s a 558C in 30 ml p e-
hyb idiza ion solu ion (1 m/ % BSA, 0.5 M Na
2
HPO
4
,
7 m/ % SDS, 1 mM EDTA, 10 mg/ml salmon spe m
DNA), and was hyb idized o 15 hou s wi h single-
s and-specific p obes o he DNA gene clus e . The
ds PCR p oduc o 1405 bp leng h, syn hesized using he
p1F and he p1R p ime pai (Supplemen a y Da a), was
used as empla e DNA o subsequen p obe p epa a ion.
Labeling wi h
32
P was pe o med ei he by andom p ime
labeling (ds p obe; using a RediP ime Ki , Ame sham) o
‘linea amplifica ion’ (using a single p ime ) o p epa e
s and-specific p obes: The p1F p ime alone was applied
o sense-specific, and he p1R p ime o an isense s and-
specific p obe p epa a ion. In hese eac ions 2.5 U Taq
polyme ase (Fe men as Li e Science, Ma yland, USA)
was used, in 50 mlo 1 eac ion buffe (10 mM T is–
HCl, 50 mM KCl, 0.08% N P-40, pH 8.8) supplemen ed
wi h 3 mM MgCl
2
, con aining 50 ng empla e DNA,
20 pmol o p ime and he nucleoside- iphospha es.
dATP, dTTP and dGTP we e used a 0.25 mM, dCTP
a 5 mM concen a ion (all om P omega Li e Science,
Madison, USA), and o each labeling eac ion 5 ml
[a
32
P]-dCTP (6000 Ci/mmol, 10 mCi/ml; Ins i u e o
Iso opes LTD, Hunga y) was added. In he fi s eac ion
cycle, dena u a ion was a 948C o 3 min, annealing a
608C o 1.5 min, polyme iza ion a 728C o 1.5 min;
his was ollowed by 45 cycles when dena u a ion was a
948C o 1.5 min, annealing a 608C o 50 s, polyme iza-
ion a 728C o 1.5 min. The p obes we e pu ified on
Sephadex G-25 (Ame sham). A e hyb idiza ion, he
memb anes we e washed h ee imes a 608C wi h a wash-
ing solu ion (40 mM Na
2
HPO
4
, 1 m/ % SDS, 1 mM
EDTA). The signal was de ec ed by Phospho-sc een
(Kodak) and isualized by a BIO-RAD Phospho-Image .
Pho og aphic equipmen and se ings
Gel pho os we e aken by FinePix S602Zoom digi al
came a (Fujifilm) and p epa ed o publica ion using
Pain Shop P o 9.0.
RESULTS AND DISCUSSION
The soli a y bands o ds PCR agmen s a e sepa a ed
in o wo dis inc bands upon u ea/hea dena u a ion and
aga ose gel elec opho esis, in he p esence o 1 M u ea, in
a size ange o 1000–10 000 nucleo ides (Figu e 1). The ss
DNA agmen s in bo h bands un much as e han he ds
DNA hey a e de i ed om. (‘ss’ s ands he e o a single
polynucleo ide chain ha has been sepa a ed om i s
complemen a y s and upon hea dena u a ion, ega dless
2Nucleic Acids Resea ch, 2009
o he likely p esence o s able o ansien ds egions in
i , while ‘ds’ is used o designa e he non-hea -dena u ed
molecula species which may become pa ially ss while
unning in he u ea–aga ose gel.) Sou he n hyb idiza ion
using ss-specific p obes e eals ha he wo bands co e-
spond o he complemen a y s ands o he ds agmen s.
The p esence o aces o iso ope-labeled molecules
ecognizing he o he s and allowed isualiza ion o
bo h s ands. The mino band co esponding o he ds
molecules in dena u ed samples (see Figu e 1B and C) is
appa en ly due o eassocia ion o he sepa a ed comple-
men a y s ands. In line wi h his, Figu e 2 demons a es
ha a e sepa a ion, he sense s and (co-linea wi h he
coding s and o he 18S, 5.8S and 25S DNA genes) and
he an isense s and o he excised bands eadily pai wi h
each o he , egene a ing he o iginal ds agmen s (see
Figu e 2B, lane 5). The wo s ands main ain hei diffe -
en ial mig a ion p ope ies e en a e e- unning hem,
sepa a ely, in aga ose de oid o u ea (Figu e 2B, lanes
3–4), sugges ing ha u ea is necessa y o sepa a e he
complemen a y s ands upon hea dena u a ion, and i s
con inued p esence in he gel sys em is no equi ed o
hei diffe en ial elec opho e ic mig a ion. A pa ial asso-
cia ion be ween ss agmen s o iden ical pola i y is also
isible in Figu e 2B, lane 4 (especially a e p olonged
incuba ion; da a no shown); hese sense–sense and
an isense–an isense complexes un simila ly o he ds ag-
men s composed o complemen a y s ands. The p esence
o nume ous hai pins, pseudokno s and en anglemen s in
he olded single s ands (simula ed using he Kine old
p og am: h p://kine old.cu ie. /cgi-bin/ o m.pl) and
he s uc u es de i ed in molecula dynamics simula ions
o ss oligonucleo ides (12) a e in line wi h he possibili y
ha such complexes may a ise when wo iden ical mole-
cules in e ac . As Figu e 3 shows, s andedness-dependen
Figu e 1. U ea–aga ose gel elec opho e ic analysis o ds and u ea/hea -dena u ed ss agmen s o a ying leng h. The ds DNA agmen s we e
p epa ed by PCR, using S. ce e isiae DNA as empla e and a se o p ime s (see ‘Ma e ials and Me hods’ sec ion) designed o yield PCR p oduc s
o 1405, 4182, 5664, 7326 and 8573-bp leng h (o e lapping a he 30-end defined by he e e se p ime ). (A) EB -s ained u ea–aga ose gel. Lanes
labeled ‘N’ and ‘D’ con ain undena u ed and dena u ed PCR p oduc s o inc easing leng h, espec i ely. The numbe s indica e he size o he PCR
p oduc s analyzed in ha lane; ‘mix’: mix u e o he PCR p oduc s. M: undena u ed 1 kb ma ke . (B, C) Sou he n blo o he gel in panel A, using a
sense-s and-specific (B) o an an isense-s and-specific (C) p obe (p epa ed as desc ibed in ‘Ma e ials and Me hods’ sec ion).
Figu e 2. Reassocia ion o he complemen a y s ands. (A) U ea–
aga ose gel elec opho e ic analysis o he 8573 bp PCR p oduc .
Lanes 1–2 (duplica e sample): undena u ed PCR p oduc s; lanes 3, 4
(duplica e sample): sense (s) and an isense (as) s and, un a e hea
dena u a ion. M: 1 kb ds ma ke . The gel was washed in 1TAE,
ena u ed in 100 mM NaCl solu ion and s ained wi h 0.5 mg/ml EB .
The bands, al eady de oid o u ea, con aining he ds PCR p oduc and
he wo complemen a y s ands we e excised and e- un on a s anda d
aga ose gel, as shown in panel B. (B) Non-dena u ing gel elec opho e-
ic analysis. Lane 1, and lanes 3–4: 8573 bp ds PCR p oduc and he
sepa a ed complemen a y s ands, espec i ely, cu ou om he fi s
gel ( om lane 1 and 3, espec i ely) and e- un di ec ly wi hou hea
dena u a ion. Lane 2: he excised block o he ds agmen s o panel A
lane 2 was hea dena u ed hen allowed o ena u e (as desc ibed in
‘Ma e ials and Me hods’ sec ion), and loaded on he gel. Lane 5: he
samples o he excised aga ose blocks con aining he complemen a y
s ands (bands labeled ‘s’ and ‘as’ in lane 4 o panel A) we e uni ed,
hea dena u ed, hen allowed o ena u e (as desc ibed in ‘Ma e ials
and Me hods’ sec ion), o allow eassocia ion o he wo s ands
be o e analysis. M: 1 kb ladde , analyzed wi hou hea dena u a ion
(bo h panels).
Nucleic Acids Resea ch, 2009 3
elec opho e ic sepa a ion is no es ic ed o pa icula
DNA sequences. The complemen a y s ands o he 
DNA HindIII agmen s a e also sepa a ed, pa icula ly
in he case o he 23 kb agmen . The iden i iy o he wo
s ands was de e mined using single-s and-specific p obes
(see Supplemen a y Da a, Figu e S1). In S. ce e isiae
genomic DNA he DNA clus e , i.e. he empla e used
o he PCR eac ions (Figu es 1 and 2), con ains epe i-
i e 9.1 kb segmen s ha can be excised wi h SmaI. This
‘in i o amplified’ DNA is also sepa a ed in o diffe en ially
mig a ing complemen a y s ands (Figu e 3A and B, lanes
3–4). The iden i y o he wo complemen a y s ands was
confi med based on he dec eased in ensi y o he band
co esponding o he an isense s and a e diges ion o
he aga ose-embedded DNA wi h a nickase enzyme
(Nb.Bpu10I) specific o his s and (Figu e 3A and B,
lanes 3–4). The size o he new ds agmen o med upon
S1 diges ion o he Nb.Bpu10I-p e ea ed blocks
(Figu e 3, lane 5) is in line wi h he (pa ial) nickase ac i -
i y expec ed. (The nicking cha ac e o his enzyme could
be demons a ed by hyb idiza ion using s and-specific
p obes, as shown in Supplemen a y Figu e 3, and by
ansla ing he nicks ou o hei specific si e using DNA
polyme ase I and nucleo ide iphospha es; da a no
shown.) Compa ison o he nucleo ide composi ion o
he opposing s ands o all he DNA agmen s analyzed
abo e e eals ha hei sepa a ion depends bo h on he
C/G and (A+T)/(C+G) a ios (Supplemen a y da a).
When he la e a io is simila , he highe he C/G
a io, he mo e e a ded he elec opho e ic mobili y o
an ss agmen will be.
In con as wi h he esul s ob ained wi h aga ose
gels, he elec opho e ic mobili y o he wo s ands was
indis inguishable in u ea–polyac ylamide gels (da a no
shown); hus he effec desc ibed he ein has no ele ance
o he wo-dimensional s andedness-dependen elec o-
pho esis app oach (17), which is based on PAGE. The
possible easons o he diffe ence be ween he wo gel
sys ems may be ela ed o (i) aga ose-specific chemical
in e ac ions wi h ss DNA, (ii) he diffe en a e age po e
size o he wo gel ypes (see abo e) o (iii) a chi ec u al
diffe ences in he gel ma ix. The e a da ion o he ds
agmen s ela i e o he ss molecules in TAE-buffe ed
gels was la ge a lowe aga ose concen a ion (da a no
shown), unde lining he ole o sie ing mechanism in di -
e en ia ing be ween he wo s ands. In e es ingly, when
un in TBE buffe ins ead o TAE (used h oughou he
expe imen s shown), he dena u ed DNA agmen s gen-
e ally mig a e slowe han he co esponding ds DNA
in 1.0–1.6% aga ose gels [da a in acco dance wi h (14);
no shown].
U ea is expec ed o comple ely dep o einize ch oma in
samples in aga ose blocks (18) and is also known o educe
T
m
by app oxima ely 3 K pe mola u ea added, indepen-
den om base composi ion (19). I seems ha 8 M u ea is
no sufficien o pe manen ly o comple ely mel duplex
egions, as aised in e . (20). This conclusion is in line
wi h he p esence o a mino band con aining undena u ed
agmen s in Figu e 2A, lanes 3–4. I is also in ag eemen
wi h he ac ha he pu ified wo s ands eadily eassoci-
a e (Figu e 2B, lane 5), and also wi h ou u ile effo s o
keep homopolyme s o poly(dG)–poly(dC) dena u ed
while unning hese samples in u ea–aga ose gels (da a
no shown). The ac ha he diffe en ial mobili y o he
wo s ands is main ained in aga ose gels de oid o u ea
(Figu e 2B, lanes 3–4) a gues agains he possibili y ha
masses o bound u ea would significan ly con ibu e o
hese effec s. Diffe en base composi ion on i s own may
esul in diffe en o e all con o ma ional cha ac e is ics.
On he o he hand, a dynamic in e ac ion be ween
he bases and he dena u an could occu and influence
he con o ma ion o he wo s ands diffe en ially.
In e ac ion o u ea wi h T bases exposed upon dena u a-
ion has been in oked om he modynamic conside a-
ions (10,19); H-bonding be ween u ea and he o he
h ee bases is also possible (19). In he p esence o u ea,
in as and pai ing is no expec ed o be hampe ed mo e
han eassocia ion o he opposing s ands ha ce ainly
akes place e en in he p esence o u ea (see Figu e 2B,
lane 5), so diffe ences in base composi ion may lead o
diffe en 3D s uc u es and diffe en mobili y e en in he
p esence o he dena u ing agen . The mobili y o ds DNA
Figu e 3. U ea–aga ose gel elec opho e ic analysis o HindIII ag-
men s and aga ose-embedded S. ce e isiae genomic DNA. (A) EB -
s ained u ea–aga ose gel. Lanes 1 and 3: SmaI-diges ed S. ce e isiae
genomic DNA, undena u ed and dena u ed, espec i ely. Lanes 2 and
4: SmaI- and Nb.Bpu10I-diges ed S. ce e isiae genomic DNA, undena-
u ed and dena u ed, espec i ely. Lane 5: SmaI- and Nb.Bpu10I-
diges ed S. ce e isiae genomic DNA, un wi hou dena u a ion bu
a e S1 nuclease diges ion. Res ic ion enzyme diges ions we e pa ial
in he aga ose blocks. M and D: HindIII agmen s wi hou and
a e hea dena u a ion, espec i ely. The numbe s on he le side o
he panel indica e he size o he Hind III ds agmen s in bp. The
diffe en size ds agmen s ma ch he diffe en ss agmen pai s as
indica ed by he connec ing lines in he figu e, e ified by e unning
in dena u ing condi ions o indi idual ds  agmen s isola ed om a
s anda d aga ose gel. The a ows poin a he 2 mplasmid also p esen
in he yeas nuclei. (B) Sou he n blo o panel A, hyb idized wi h a ds
p obe hyb idizing equally wi h he sense and an isense DNA s ands
(p epa ed as desc ibed in ‘Ma e ials and Me hods’ sec ion). The bands
co esponding o he ds and dena u ed, ss 9.1 kb genomic DNA epea
a e indica ed. The iden i y o he wo complemen a y s ands was con-
fi med based on he dec eased in ensi y o he band co esponding
o he an isense s and a e diges ion o he aga ose-embedded DNA
wi h a nickase enzyme (Nb.Bpu10I) specific o his s and (lanes 3–4).
The size o he new ds agmen o med upon S1 diges ion o he
Nb.Bpu10I-p e ea ed blocks (lane 5) is in line wi h he nickase ac i i y
expec ed.
4Nucleic Acids Resea ch, 2009
in gel elec opho esis is p ima ily dependen on s and
size, and o a much lesse ex en , he pa icula nucleo ide
sequence (21). The mobili y o single s ands, howe e ,
is conside ably influenced by small changes in sequence.
The sequence sensi i i y o he 3D s uc u es o med is he
basis o he single-s and con o ma ion polymo phism
(SSCP) echnologies applicable in he 300 bp ange
(22). Based on he abo e conside a ions, and in iew o
he simila in e p e a ion o SSCP phenomena in he sho
agmen anges, sequence-dependen con o ma ional di -
e ences be ween he opposi e s ands a e p oposed o
explain hei diffe en ial mig a ion in u ea–aga ose gels.
Analogous obse a ions using alkaline dena u a ion
ha e also been epo ed wi hou being cha ac e ized
wi h he pu pose o gene al applicabili y (23,24). The
no el applica ion o u ea–aga ose gel elec opho esis is
p ima ily ecommended as an easy way o p epa e ss
p obes. In addi ion, i offe s a simple p ocedu e o he
s and-specific analysis o CpG me hyla ion, discon inu-
i ies (including nicks) p esen in he DNA s ands (see
Figu e 3, lanes 4–5) and sepa a ion o diffe en ially labeled
wo s ands o subsequen analysis. Ou me hod is no
influenced by he p esence o alkali-sensi i e si es and i
can be u ilized in a ious cell biological esea ch a eas,
including he dis inc mechanisms o leading and lagging
s and DNA syn hesis, he puzzle o ‘immo al s and’
hypo hesis (25), analysis o s and-specific epai p o-
cesses, he mechanism o imp in ing leading o he
ma ing- ype swi ch in yeas ( ha was a ibu ed o a
lagging-s and-specific nick gene a ed by unknown
mechanism) (26–29), he analysis o opoisome ase-
media ed clea ages and he mechanism in ol ed in he
gene a ion o nicks upon class swi ch ecombina ion a
he immunoglobulin hea y chain egion (30).
SUPPLEMENTARY DATA
Supplemen a y Da a a e a ailable a NAR Online.
ACKNOWLEDGEMENTS
The au ho s hank Pal Ge gely (MHSC, Deb ecen) o
aluable ad ice ega ding he mechanism o diffe en ial
mobili y o he wo s ands, Pe e Hajdu (MHSC,
Deb ecen) o me hodical ad ice and Wol -Die ich
Heye (Sec ion o Mic obiology, Uni e si y o Cali o nia,
Da is, USA) o he WDHY199 s ain o S. ce e isiae and
Gyongyi Dajka o excellen echnical assis ance.
FUNDING
The Hunga ian Scien ific Resea ch Fund (OTKA)
[TO48742, 72762 o G. Sz.]; he Minis y o Public
Heal h ETT [067/2006 o G. Sz.]; Janos Bolyai esea ch
und o he Hunga ian Academy o Sciences ( o E.K.).
Funding o open access cha ge: OTKA.
Conflic o in e es s a emen . None decla ed.
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