Sequential DNA hybridisation assays by fast micromixing.
Identifieur interne : 003039 ( Main/Exploration ); précédent : 003038; suivant : 003040Sequential DNA hybridisation assays by fast micromixing.
Auteurs : Martin Heule [Suisse] ; Andreas ManzSource :
- Lab on a chip [ 1473-0197 ] ; 2004.
Descripteurs français
- KwdFr :
- MESH :
English descriptors
- KwdEn :
- MESH :
- chemical , chemistry : DNA.
- instrumentation : Microfluidics.
- methods : Microfluidics.
- Equipment Design, Kinetics, Nucleic Acid Hybridization, Polymorphism, Single Nucleotide, Time Factors.
Abstract
The prospects of performing DNA hybridisation assays in a novel sequential scheme are explored in this article. It is based on recording the kinetics of hybridisation on a microfluidic device measuring only 10 by 5 mm. It contains a split channel system for fast mixing and a subsequent meandering channel to observe the evolution of the mixture by optical means. The problems of diffusion limitations in the laminar flow regime are overcome by reducing the average diffusion distance to a few micrometers only. DNA oligomers (20-mers) of different sequences were injected on the chip for mixing. The detection of hybridisation was based on the fluorescence of DNA-intercalating dyes. Two modes of operation were investigated. First, the samples were injected into the micromixing device at a high flow rate of 40 microl min(-1). When the sample passed through the actual micromixing unit, the flow rate was reduced to allow for measurement of fluorescence levels at various steady-state reaction times in the range of 2-15 s, as defined by the channel geometry. Using this continuous flow approach, photobleaching of fluorophores could be avoided. In a buffer containing 0.2 M NaCl, 2 base-pair mismatches could routinely be detected within 5-20 s. Single base-pair mismatches were successfully identified under low salt conditions. In the second mode, the flow was completely stopped and the evolution of the total fluorescence signal influenced by the hybridisation of oligomers and photobleaching was observed. Whereas the sequence-dependent effects remained unchanged, the assay times between the mixing of two oligomers and clear identification of their hybridisation properties could be reduced down to a maximum of 5-7 s, in some cases even below 1 s.
DOI: 10.1039/b404633a
PubMed: 15472736
Affiliations:
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Le document en format XML
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Chemistry, London, UKSW7 2AY. m.heule@hispeed.ch</nlm:affiliation><country wicri:rule="url">Suisse</country></affiliation></author><author><name sortKey="Manz, Andreas" sort="Manz, Andreas" uniqKey="Manz A" first="Andreas" last="Manz">Andreas Manz</name></author></analytic><series><title level="j">Lab on a chip</title><idno type="ISSN">1473-0197</idno><imprint><date when="2004" type="published">2004</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>DNA (chemistry)</term><term>Equipment Design</term><term>Kinetics</term><term>Microfluidics (instrumentation)</term><term>Microfluidics (methods)</term><term>Nucleic Acid Hybridization</term><term>Polymorphism, Single Nucleotide</term><term>Time Factors</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ADN ()</term><term>Cinétique</term><term>Conception d'appareillage</term><term>Facteurs temps</term><term>Hybridation d'acides nucléiques</term><term>Microfluidique ()</term><term>Microfluidique (instrumentation)</term><term>Polymorphisme de nucléotide simple</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>DNA</term></keywords><keywords scheme="MESH" qualifier="instrumentation" xml:lang="en"><term>Microfluidics</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Microfluidics</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Equipment Design</term><term>Kinetics</term><term>Nucleic Acid Hybridization</term><term>Polymorphism, Single Nucleotide</term><term>Time Factors</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>ADN</term><term>Cinétique</term><term>Conception d'appareillage</term><term>Facteurs temps</term><term>Hybridation d'acides nucléiques</term><term>Microfluidique</term><term>Polymorphisme de nucléotide simple</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The prospects of performing DNA hybridisation assays in a novel sequential scheme are explored in this article. It is based on recording the kinetics of hybridisation on a microfluidic device measuring only 10 by 5 mm. It contains a split channel system for fast mixing and a subsequent meandering channel to observe the evolution of the mixture by optical means. The problems of diffusion limitations in the laminar flow regime are overcome by reducing the average diffusion distance to a few micrometers only. DNA oligomers (20-mers) of different sequences were injected on the chip for mixing. The detection of hybridisation was based on the fluorescence of DNA-intercalating dyes. Two modes of operation were investigated. First, the samples were injected into the micromixing device at a high flow rate of 40 microl min(-1). When the sample passed through the actual micromixing unit, the flow rate was reduced to allow for measurement of fluorescence levels at various steady-state reaction times in the range of 2-15 s, as defined by the channel geometry. Using this continuous flow approach, photobleaching of fluorophores could be avoided. In a buffer containing 0.2 M NaCl, 2 base-pair mismatches could routinely be detected within 5-20 s. Single base-pair mismatches were successfully identified under low salt conditions. In the second mode, the flow was completely stopped and the evolution of the total fluorescence signal influenced by the hybridisation of oligomers and photobleaching was observed. Whereas the sequence-dependent effects remained unchanged, the assay times between the mixing of two oligomers and clear identification of their hybridisation properties could be reduced down to a maximum of 5-7 s, in some cases even below 1 s.</div></front></TEI><affiliations><list><country><li>Suisse</li></country></list><tree><noCountry><name sortKey="Manz, Andreas" sort="Manz, Andreas" uniqKey="Manz A" first="Andreas" last="Manz">Andreas Manz</name></noCountry><country name="Suisse"><noRegion><name sortKey="Heule, Martin" sort="Heule, Martin" uniqKey="Heule M" first="Martin" last="Heule">Martin Heule</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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