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Single-nucleotide polymorphism analysis by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device
KTH, Superseded Departments, Signals, Sensors and Systems.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Signals, Sensors and Systems.
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0002-4657-8532
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2003 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 24, no 1-2, 158-161 p.Article in journal (Refereed) Published
Abstract [en]

We describe a microfluidic approach for allele-specific extension of fluorescently labeled nucleotides for scoring of single-nucleotide polymorphism (SNP). The method takes advantage of the fact that the reaction kinetics differs between matched and mismatched configurations of allele-specific primers hybridized to DNA template. A microfluidic flow-through device for biochemical reactions on beads was used to take advantage of the reaction kinetics to increase the sequence specificity of the DNA polymerase, discriminating mismatched configurations from matched. The volume of the reaction chamber was 12.5 nL. All three possible variants of an SNP site at codon 72 of the p53 gene were scored using our approach. This work demonstrates the possibility of scoring SNP by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device, The sensitive detection system and easy microfabrication of the microfluidic device enable further miniaturization and production of an array format of microfluidic devices for high-throughput SNP analysis.

Place, publisher, year, edition, pages
2003. Vol. 24, no 1-2, 158-161 p.
Keyword [en]
filter chamber, flow-through, fluorescence, microfluidics, single-nucleotide polymorphism
National Category
Medical Genetics
Identifiers
URN: urn:nbn:se:kth:diva-5008DOI: 10.1002/elps.200390008ISI: 000180637300021PubMedID: 12652586OAI: oai:DiVA.org:kth-5008DiVA: diva2:7462
Note
QC 20100929 NR 20140805Available from: 2005-04-01 Created: 2005-04-01 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Microfluidic bead-based methods for DNA analysis
Open this publication in new window or tab >>Microfluidic bead-based methods for DNA analysis
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

With the completion of the human genome sequencing project, attention is currently shifting toward understanding how genetic variation, such as single nucleotide polymorphism (SNP), leads to disease. To identify, understand, and control biological mechanisms of living organisms, the enormous amounts of accumulated sequence information must be coupled to faster, cheaper, and more powerful technologies for DNA, RNA, and protein analysis. One approach is the miniaturization of analytical methods through the application of microfluidics, which involves the manipulation of fluids in micrometer-sized channels. Advances in microfluidic chip technology are expected to play a major role in the development of cost-effective and rapid DNA analysis methods.

This thesis presents microfluidic approaches for different DNA genotyping assays. The overall goal is to combine the potential of the microfluidic lab-on-a-chip concept with biochemistry to develop and improve current methods for SNP genotyping. Three genotyping assays using miniaturized microfluidic approaches are addressed.

The first two assays are based on primer extension by DNA polymerase. A microfluidic device consisting of a flow-through filter chamber for handling beads with nanoliter liquid volumes was used in these studies. The first assay involved an allelespecific extension strategy. The microfluidic approach took advantage of the different reaction kinetics of matched and mismatched configurations at the 3’-ends of a primer/template complex. The second assay consisted of adapting pyrosequencing technology, a bioluminometric DNA sequencing assay based on sequencing-bysynthesis, to a microfluidic flow-through platform. Base-by-base sequencing was performed in a microfluidic device to obtain accurate SNP scoring data on nanoliter volumes. This thesis also presents the applications of monolayer of beads immobilized by microcontact printing for chip-based DNA analysis. Single-base incorporation could be detected with pyrosequencing chemistry on these monolayers.

The third assay developed is based on a hybridization technology termed Dynamic Allele-Specific Hybridization (DASH). In this approach, monolayered beads containing DNA duplexes were randomly immobilized on the surface of a microheater chip. DNA melting-curve analysis was performed by dynamically heating the chip while

simultaneously monitoring the DNA denaturation profile to determine the genotype. Multiplexing based on single-bead analysis was achieved at heating rates more than 20 times faster than conventional DASH provides.

Place, publisher, year, edition, pages
Stockholm: KTH, 2005. x, 52 p.
Series
Trita-ILA, ISSN 0281-2878 ; 0502
Keyword
Genetics, single nucleotide polymorphism, DNA analysis, SNP, microfluidics, pyrosequencing, beads, lab on a chip, hybridization, DASH, microsystem, micro totat analysis system, allele-specific extension, DASH, microcontact printing, Genetik
National Category
Medical Genetics
Identifiers
urn:nbn:se:kth:diva-155 (URN)91-7283-992-9 (ISBN)
Public defence
2005-04-08, Q2, Osquldas v 10, KTH, 13:00
Opponent
Note
QC 20101008Available from: 2005-04-01 Created: 2005-04-01 Last updated: 2010-10-08Bibliographically approved

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