Notes &Tips
Analysis of supercoiled DNA by agaro gel electrophoresis using low-conducting sodium threonine medium
Tomomi Ishido,Mitsuru Ishikawa,Ken Hirano *
Health Technology Rearch Center,National Institute of Advanced Industrial Science and Technology (AIST),Takamatsu,Kagawa 761-0395,Japan
a r t i c l e i n f o Article history:
Received 3December 2009
Received in revid form 9January 2010Accepted 14January 2010
Available online 18January 2010
a b s t r a c t凉反义词
足浴盘We describe a new low-ionic-strength sodium threonine (STh)medium with the advantage of avoidin
g relative DNA band migration changes following electrophoresis of supercoiled DNA in agaro gel when substituted for the standard conductive medium of TBE (Tris–boric acid–ethylenediaminetetraacetic acid [EDTA])or TAE (Tris–acetic acid–EDTA)or the low-ionic-strength sodium boric acid medium.Low-ionic-strength STh medium provided better resolution,less heat generation,and prevention of relative migra-tion order changes among linear,covalently clod circular-,and open circular-formed DNA in the range of 2–10kiloba pairs in 1%agaro gel electrophoresis.
妙招治失眠Ó2010Elvier Inc.All rights rerved.
Plasmid DNA has been widely ud not only as a genetic vector in recombinant technology but also as a substrate for various anal-ys such as tho of topological mechanisms by topoisomeras [1,2],DNA strand damage by ultraviolet (UV)1radiation,metal ions and free radicals [3,4],and anticancer and antibacterial drugs [5,6].The plasmid DNAs for the studies are usually analyzed by electro-phoresis,leading to differential migration of forms of supercoiled plasmid that differ in topology:covalently clod circular (ccc,form I),open circular (oc,form II),and linear DNA (form III).The electro-phoresis is usually performed in standard conductive medium of TAE (Tris–acetic acid–ethylenediaminetetraacetic acid [EDTA])or TBE (Tris–boric acid–EDTA),each of which has the
disadvantage that high voltages cannot be applied due to the high ionic strength of the medium [7].The novel low-ionic-strength sodium boric acid (SB)medium was recently shown to decrea the running time and improve the resolution of DNA fragments in agaro gel [8]and polyacrylamide gel systems [9].Thus,SB medium is now widely ud in molecular biology,medical diagnosis,forensics,and micro-bial identification.However,information is lacking on the electro-phoretic behavior of supercoiled DNA using low-ionic-strength medium.In the current study,we found that SB medium dramati-cally lowers electrophoretic migration of cccDNA and ocDNA when compared with TAE and TBE media.This phenomenon of decread migration complicates analysis of plasmids due to the changed rela-tionship between linear and circular (ccc and oc)DNA migration,and
it gives long running times and low resolution in electrophoresis.Furthermore,standard TBE medium leads to low resolution in the range of 5kiloba pairs (kbp)or more.In the current study,we ud a new low-ionic-strength sodium threonine (STh)medium and eval-uated tolerance for migration order changes as well as the DNA band resolution compared with standard and SB media for rapid agaro gel electrophoretic analysis of differing forms of supercoiled plasmid DNA.
All DNA samples (0.5l g/lane)were analyzed by agaro gel electrophoresis using the Mini Sub-Cell
GT system (Bio-Rad,Her-cules,CA,USA)at 150V for 40min.Gels were 1.0%agaro (Sea-Kem GTG Agaro,Lonza Rockland,Bal,Switzerland)and 10cm long.Current and temperature were recorded at the start and end of electrophoresis.After electrophoresis,DNA bands were stained and visualized with ethidium bromide (Bio-Rad).The STh medium was prepared at a final concentration of 17mM sodium hydroxide (Nacalai Tesque,Kyoto,Japan)and 39mM threonine (ICN Biomedicals,Aurora,OH,USA)at pH 9.0.Comparison media were prepared of SB (10mM sodium hydroxide,pH 8.5,adjusted with boric acid),TBE (89mM Tris,89mM boric acid,and 2mM EDTA,pH 8.2),and TAE (40mM Tris,20mM acetic acid,and 1mM EDTA,pH 8.0).A 1-kbp DNA ladder as the linear DNA and a supercoiled DNA ladder as the cccDNA were purchad from New England Biolabs (Ipswich,MA,USA).The ocDNA ladder was prepared (unwound)from the supercoiled DNA ladder using the nick-translation method slightly modified by substituting a mix-ture of analog and natural deoxynucleoside 50-triphosphates (dNTPs)for natural dNTPs.The reaction mixture (20l l)containing 0.6l g of supercoiled DNA ladder,20l M of each dNTP,an enzyme mixture of DNa I and DNA polymera I,and reaction buffer was incubated at 15°C for 35min.The enzyme mixture and reaction
0003-2697/$-e front matter Ó2010Elvier Inc.All rights rerved.doi:10.1016/j.ab.2010.01.020
*Corresponding author.Fax:+81878693569.E-mail address:jp (K.Hirano).1
Abbreviations ud:UV,ultraviolet;ccc,covalently clod circular;oc,open circular;TAE,Tris–acetic acid–ethylenediaminetetraacetic acid;EDTA,ethylenedi-aminetetraacetic acid;TBE,Tris–boric acid–EDTA;SB,sodium boric acid;kbp,kiloba pairs;STh,sodium threonine;dNTP,deoxynucleoside 5’-triphosphate.Analytical Biochemistry 400(2010)
148–150
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buffer were included in the nick-translation kit (Roche,Mannheim,Germany).The unwinding reaction was quenched by heating at 65°C for 10min.
Fig.1shows analysis of linear DNA,cccDNA,and ocDNA (2–10kbp)under four conductive media (STh,SB,TBE,and TAE)in 1%agaro gel electrophoresis.It is known that migration of super-coiled DNA of a given molecular weight is usually in the order of cccDNA first,followed by linear DNA and then ocDNA,although the migration depends on buffer,agaro concentration,and elec-trophoresis conditions [10].U of SB or TBE medium,however,dramatically altered the migration order,especiall
y for cccDNA.SB medium decread migration of cccDNA and ocDNA compared with that of linear DNA in all size ranges of 2–10kbp.In TBE med-ium,the migration order depended on the DNA size.The migra-tion changes significantly complicate the analysis of supercoiled DNA such as plasmids.Furthermore,the ocDNA larger than 5kbp in SB and TBE media was not resolved.
On the other hand,STh medium not only gave excellent results in parating and distinguishing supercoiled DNA but also showed tolerance for any migration order changes in the range of 2–10kbp compared with SB and TBE media (Fig.1).TAE med-ium provided similar migration results;however,resolution be-tween linear DNA and cccDNA was slightly lower than that in STh medium.The 10-kbp ocDNA band in TAE was difficult to dis-tinguish becau the band was substantially smeared in our experiment for unknown reasons.Furthermore,during electro-phoresis the current and temperature increas in TAE medium were higher than (approximately twice)tho in STh medium,with the current for TAE increasing from 128to 186mA and the temperature rising by 20°C.By contrast,the corresponding increas in low-ionic-strength STh medium (17mM sodium)were from 98to 122mA and 10°C.STh medium made current stable and low per unit voltage,resulting in less heat generation.Thus,STh medium may provide not only tolerance for migration order changes but also rapid paration due to its low ionic strength.
We also explored low-ionic-strength,sodium-bad conduc-tive media and tried various medium components (e.g.,veral esntial amino acids).However,STh provided the clearest reso-lution while also having low ionic strength.The reason why STh gives high resolution is not well understood at this time.How-
ever,the amino acids containing carboxylic or hydroxyl side chains (threonine,rine,glutamic acid,and aspartic acid)tended to yield high resolution compared with other amino acids (data not shown).Moreover,the mobility of cccDNA and ocDNA was greater than that of linear DNA,depending on the sodium concen-tration (data not shown).The threshold concentration of sodium in STh medium,to provide migration order consistency for super-coiled DNA,was 17mM.
In conclusion,we found that in 1%agaro gel electrophoresis,low-ionic-strength STh medium provided excellent paration and tolerance for migration order changes of supercoiled DNA fragments in the range of 2–10kbp.The current study is impor-tant for developing a method for rapid analysis of supercoiled plasmid DNA to resolve the problem of migration order changes.SeaKem GTG agaro has been specifically designed for prepara-tive DNA electrophoresis.Therefore,despite its higher cost rela-tive to some other agaro preparations,isolation and recovery of DNA from this agaro are likely to be more efficient.With this in mind,this rapid analysis bad on agaro gel elec开展工作
trophoresis could have the advantage of cost savings per run when compared with analysis by microchip-bad electrophoresis [11]and time savings in isolation and recovery of plasmid DNA from agaro gel.
Acknowledgment
This work was supported by a grant from the Industrial Tech-nology Rearch Program,New Energy and Industrial Technology Development Organization (NEDO),Japan.References
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Fig.1.Analysis of electrophoretic migration of supercoiled DNA showing linear DNA (À),cccDNA (1),and ocDNA (s )using four conductive media (STh,SB,TBE,and TAE)by 1%agaro gel electrophoresis performed at 150V for 40min.Current and temperature were recorded at the start and end of electrophoresis.The difference between the start and end temperatures is denoted as D T.
The linear DNA ladder lanes show only 1.5,2.0,3.0,4.0,5.0,6.0,8.0,and 10.0kbp,although the ladder originally also included 0.5and 1.0kbp.The cccDNA and ocDNA ladder is a mixture of 2.0,2.5,3.0,3.5,4.0,5.0,6.0,8.0,and 10.0kbp supercoiled DNA fragments.The dotted lines indicate 2.0,3.0,5.0,and 10.0kbp for reference.Note that the dotted line between cccDNA and ocDNA at 10.0kbp in TAE is abnt becau the 10.0-kbp ocDNA band in TAE was difficult to distinguish.DNA bands were stained with ethidium bromide following electrophoresis.
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