Sequence-based separation of single-stranded DNA using nucleotides in capillary electrophoresis: focus on phosphate.
Identifieur interne : 001F12 ( Main/Exploration ); précédent : 001F11; suivant : 001F13Sequence-based separation of single-stranded DNA using nucleotides in capillary electrophoresis: focus on phosphate.
Auteurs : Xueru Zhang [États-Unis] ; Linda B. McgownSource :
- Electrophoresis [ 1522-2683 ] ; 2013.
Descripteurs français
- KwdFr :
- ADN simple brin (), ADN simple brin (isolement et purification), Concentration en ions d'hydrogène, Données de séquences moléculaires, Dénaturation d'acide nucléique, Glucides (), Guanosine monophosphate (), Humains, Substances tampon, Séquence nucléotidique, Température, Électrophorèse capillaire ().
- MESH :
English descriptors
- KwdEn :
- Base Sequence, Buffers, Carbohydrates (chemistry), DNA, Single-Stranded (chemistry), DNA, Single-Stranded (isolation & purification), Electrophoresis, Capillary (methods), Guanosine Monophosphate (chemistry), Humans, Hydrogen-Ion Concentration, Molecular Sequence Data, Nucleic Acid Denaturation, Temperature.
- MESH :
- chemical , chemistry : Carbohydrates, DNA, Single-Stranded, Guanosine Monophosphate.
- chemical , isolation & purification : DNA, Single-Stranded.
- chemical : Buffers.
- methods : Electrophoresis, Capillary.
- Base Sequence, Humans, Hydrogen-Ion Concentration, Molecular Sequence Data, Nucleic Acid Denaturation, Temperature.
Abstract
DNA analysis has widespread applicability in biology, medicine, biotechnology, and forensics. DNA separation by length is readily achieved using sieving gels in electrophoresis. Separation by sequence is less simple, generally requiring adequate differences in native or induced conformation or differences in thermal or chemical stability of the strands that are hybridized prior to measurement. We previously demonstrated separation of four single-stranded DNA 76-mers that differ by only a few A-G substitutions based solely on sequence using guanosine-5'-monophosphate (GMP) in the running buffer. We attributed separation to the unique self-assembly of GMP to form higher order structures. Here, we examine an expanded set of 76-mers designed to probe the mechanism of the separation and effects of experimental conditions. We were surprised to find that other ribonucleotides achieved the similar separation to GMP, and that some separation was achieved using sodium phosphate instead of GMP. Potassium phosphate achieved almost as good separations as the ribonucleotides. This suggests that the separation medium provides a physicochemical environment for the DNA that effects strand migration in a sequence-selective manner. Further investigation is needed to determine whether the mechanism involves specific interactions between the phosphates and the DNA strands or is a result of other properties of the separation medium. Phosphate generally has been avoided in DNA separations by capillary gel electrophoresis because its high ionic strength exacerbates Joule heating. Our results suggest that phosphate compounds should be examined for separation of DNA based on sequence.
DOI: 10.1002/elps.201200683
PubMed: 23576075
Affiliations:
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Mcgown</name></author></analytic><series><title level="j">Electrophoresis</title><idno type="eISSN">1522-2683</idno><imprint><date when="2013" type="published">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Base Sequence</term><term>Buffers</term><term>Carbohydrates (chemistry)</term><term>DNA, Single-Stranded (chemistry)</term><term>DNA, Single-Stranded (isolation & purification)</term><term>Electrophoresis, Capillary (methods)</term><term>Guanosine Monophosphate (chemistry)</term><term>Humans</term><term>Hydrogen-Ion Concentration</term><term>Molecular Sequence Data</term><term>Nucleic Acid Denaturation</term><term>Temperature</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ADN simple brin ()</term><term>ADN simple brin (isolement et purification)</term><term>Concentration en ions d'hydrogène</term><term>Données de séquences moléculaires</term><term>Dénaturation d'acide nucléique</term><term>Glucides ()</term><term>Guanosine monophosphate ()</term><term>Humains</term><term>Substances tampon</term><term>Séquence nucléotidique</term><term>Température</term><term>Électrophorèse capillaire ()</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Carbohydrates</term><term>DNA, Single-Stranded</term><term>Guanosine Monophosphate</term></keywords><keywords scheme="MESH" type="chemical" qualifier="isolation & purification" xml:lang="en"><term>DNA, Single-Stranded</term></keywords><keywords scheme="MESH" type="chemical" xml:lang="en"><term>Buffers</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>Base Sequence</term><term>Humans</term><term>Hydrogen-Ion Concentration</term><term>Molecular Sequence Data</term><term>Nucleic Acid Denaturation</term><term>Temperature</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>ADN simple brin</term><term>Concentration en ions d'hydrogène</term><term>Données de séquences moléculaires</term><term>Dénaturation d'acide nucléique</term><term>Glucides</term><term>Guanosine monophosphate</term><term>Humains</term><term>Substances tampon</term><term>Séquence nucléotidique</term><term>Température</term><term>Électrophorèse capillaire</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">DNA analysis has widespread applicability in biology, medicine, biotechnology, and forensics. DNA separation by length is readily achieved using sieving gels in electrophoresis. Separation by sequence is less simple, generally requiring adequate differences in native or induced conformation or differences in thermal or chemical stability of the strands that are hybridized prior to measurement. We previously demonstrated separation of four single-stranded DNA 76-mers that differ by only a few A-G substitutions based solely on sequence using guanosine-5'-monophosphate (GMP) in the running buffer. We attributed separation to the unique self-assembly of GMP to form higher order structures. Here, we examine an expanded set of 76-mers designed to probe the mechanism of the separation and effects of experimental conditions. We were surprised to find that other ribonucleotides achieved the similar separation to GMP, and that some separation was achieved using sodium phosphate instead of GMP. Potassium phosphate achieved almost as good separations as the ribonucleotides. This suggests that the separation medium provides a physicochemical environment for the DNA that effects strand migration in a sequence-selective manner. Further investigation is needed to determine whether the mechanism involves specific interactions between the phosphates and the DNA strands or is a result of other properties of the separation medium. Phosphate generally has been avoided in DNA separations by capillary gel electrophoresis because its high ionic strength exacerbates Joule heating. Our results suggest that phosphate compounds should be examined for separation of DNA based on sequence.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>État de New York</li></region></list><tree><noCountry><name sortKey="Mcgown, Linda B" sort="Mcgown, Linda B" uniqKey="Mcgown L" first="Linda B" last="Mcgown">Linda B. Mcgown</name></noCountry><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></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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