Sequence-based separation of single-stranded DNA at high salt concentrations in capillary zone electrophoresis.
Identifieur interne : 001024 ( Main/Exploration ); précédent : 001023; suivant : 001025Sequence-based separation of single-stranded DNA at high salt concentrations in capillary zone electrophoresis.
Auteurs : Xueru Zhang [États-Unis] ; Linda B. Mcgown [États-Unis]Source :
- Electrophoresis [ 1522-2683 ] ; 2016.
Descripteurs français
- KwdFr :
- MESH :
- isolement et purification : ADN simple brin.
- Colorants fluorescents, Dichroïsme circulaire, Sels, Électrophorèse capillaire.
English descriptors
- KwdEn :
- MESH :
- chemical , chemistry : Fluorescent Dyes, Salts.
- chemical , isolation & purification : DNA, Single-Stranded.
- methods : Electrophoresis, Capillary.
- Circular Dichroism.
Abstract
DNA separation by fragment length can be readily achieved using sieving gels in electrophoresis. Separation by sequence has not been as simple, generally requiring adequate differences in native or induced conformation between single or hybridized strands or differences in thermal or chemical stability of hybridized strands. Previously, it was shown that four single-stranded DNA (ssDNA) 76-mers that differ by only a few A-G substitutions could be separated based solely on sequence by adding guanosine-5'-monophosphate to the running buffer in capillary zone electrophoresis (CZE). The separation was attributed to interactions of the ssDNA with self-assembled guanine-tetrad structures; however, subsequent studies of an expanded set of ten 76-mers showed that the separation was a more general phenomenon that occurred at high salt concentrations. With the long-term goal of using experimental and computational methods to provide insight into the basis of the separation, a set of ssDNA 15-mers was designed including a poly(dT) 15-mer and nine variants. Separations were performed using fluorescent-labeled ssDNA in CZE with laser-induced fluorescence detection. Results show that separation improves with increasing buffer concentration and decreasing temperature, due at least in part to longer separation times. Migration times increase with increasing purine content, with A having a much larger effect that G. Circular dichroism spectra of the mixtures of the strands suggest that the separation is not due to changes in conformation of the ssDNA at high salt concentrations.
DOI: 10.1002/elps.201600138
PubMed: 27189739
Affiliations:
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Mcgown</name><affiliation wicri:level="2"><nlm:affiliation>Department of Chemistry and Chemical Biology Rensselaer Polytechnic Institute, Troy, NY, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Chemistry and Chemical Biology Rensselaer Polytechnic Institute, Troy, NY</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2016">2016</date><idno type="RBID">pubmed:27189739</idno><idno type="pmid">27189739</idno><idno type="doi">10.1002/elps.201600138</idno><idno type="wicri:Area/PubMed/Corpus">001116</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">001116</idno><idno type="wicri:Area/PubMed/Curation">001116</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Curation">001116</idno><idno type="wicri:Area/PubMed/Checkpoint">000F03</idno><idno type="wicri:explorRef" wicri:stream="Checkpoint" wicri:step="PubMed">000F03</idno><idno type="wicri:Area/Ncbi/Merge">001629</idno><idno type="wicri:Area/Ncbi/Curation">001629</idno><idno type="wicri:Area/Ncbi/Checkpoint">001629</idno><idno type="wicri:Area/Main/Merge">001027</idno><idno type="wicri:Area/Main/Curation">001024</idno><idno type="wicri:Area/Main/Exploration">001024</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Sequence-based separation of single-stranded DNA at high salt concentrations in capillary zone electrophoresis.</title><author><name sortKey="Zhang, Xueru" sort="Zhang, Xueru" uniqKey="Zhang X" first="Xueru" last="Zhang">Xueru Zhang</name><affiliation wicri:level="2"><nlm:affiliation>Department of Chemistry and Chemical Biology Rensselaer Polytechnic Institute, Troy, NY, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Chemistry and Chemical Biology Rensselaer Polytechnic Institute, Troy, NY</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation></author><author><name sortKey="Mcgown, Linda B" sort="Mcgown, Linda B" uniqKey="Mcgown L" first="Linda B" last="Mcgown">Linda B. Mcgown</name><affiliation wicri:level="2"><nlm:affiliation>Department of Chemistry and Chemical Biology Rensselaer Polytechnic Institute, Troy, NY, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Department of Chemistry and Chemical Biology Rensselaer Polytechnic Institute, Troy, NY</wicri:regionArea><placeName><region type="state">État de New York</region></placeName></affiliation></author></analytic><series><title level="j">Electrophoresis</title><idno type="eISSN">1522-2683</idno><imprint><date when="2016" type="published">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Circular Dichroism</term><term>DNA, Single-Stranded (isolation & purification)</term><term>Electrophoresis, Capillary (methods)</term><term>Fluorescent Dyes (chemistry)</term><term>Salts (chemistry)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ADN simple brin (isolement et purification)</term><term>Colorants fluorescents ()</term><term>Dichroïsme circulaire</term><term>Sels ()</term><term>Électrophorèse capillaire ()</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Fluorescent Dyes</term><term>Salts</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>DNA, Single-Stranded</term></keywords><keywords scheme="MESH" qualifier="isolement et purification" xml:lang="fr"><term>ADN simple brin</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Electrophoresis, Capillary</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Circular Dichroism</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Colorants fluorescents</term><term>Dichroïsme circulaire</term><term>Sels</term><term>Électrophorèse capillaire</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">DNA separation by fragment length can be readily achieved using sieving gels in electrophoresis. Separation by sequence has not been as simple, generally requiring adequate differences in native or induced conformation between single or hybridized strands or differences in thermal or chemical stability of hybridized strands. Previously, it was shown that four single-stranded DNA (ssDNA) 76-mers that differ by only a few A-G substitutions could be separated based solely on sequence by adding guanosine-5'-monophosphate to the running buffer in capillary zone electrophoresis (CZE). The separation was attributed to interactions of the ssDNA with self-assembled guanine-tetrad structures; however, subsequent studies of an expanded set of ten 76-mers showed that the separation was a more general phenomenon that occurred at high salt concentrations. With the long-term goal of using experimental and computational methods to provide insight into the basis of the separation, a set of ssDNA 15-mers was designed including a poly(dT) 15-mer and nine variants. Separations were performed using fluorescent-labeled ssDNA in CZE with laser-induced fluorescence detection. Results show that separation improves with increasing buffer concentration and decreasing temperature, due at least in part to longer separation times. Migration times increase with increasing purine content, with A having a much larger effect that G. Circular dichroism spectra of the mixtures of the strands suggest that the separation is not due to changes in conformation of the ssDNA at high salt concentrations.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>État de New York</li></region></list><tree><country name="États-Unis"><region name="État de New York"><name sortKey="Zhang, Xueru" sort="Zhang, Xueru" uniqKey="Zhang X" first="Xueru" last="Zhang">Xueru Zhang</name></region><name sortKey="Mcgown, Linda B" sort="Mcgown, Linda B" uniqKey="Mcgown L" first="Linda B" last="Mcgown">Linda B. 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