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Rapid melting curve analysis on monolayered beads for high-throughput genotyping of single-nucleotide polymorphisms
KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).ORCID iD: 0000-0002-9327-2544
KTH, School of Electrical Engineering (EES), Microsystem Technology (Changed name 20121201).
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2006 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 78, no 7, 2220-2225 p.Article in journal (Refereed) Published
Abstract [en]

This report describes a rapid solid-phase melting curve analysis method for single-nucleotide polymorphism (SNP) genotyping. The melting curve analysis is based on dynamic allele-specific hybridization (DASH). The DNA duplexes are conjugated on beads that are immobilized on the surface of a microheater chip with integrated heaters and temperature sensors. SNP on PCR products were scored, illustrating the sensitivity and robustness of the system. The method is based on random bead immobilization by microcontact printing. Single-bead detection and multiplexing were performed with a heating rate more than 20 times faster than conventional DASH. Analyses that took more than 15 min could be performed in less that 1 min, enabling ultrarapid SNP analysis. In addition, an array version of the chip was implemented enabling the preparation of an array of bead arrays for high-throughput and rapid SNP genotyping.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2006. Vol. 78, no 7, 2220-2225 p.
Keyword [en]
allele-specific hybridization, real-time, dna, system, arrays
National Category
Medical Genetics
Identifiers
URN: urn:nbn:se:kth:diva-5012DOI: 10.1021/ac051771uISI: 000236686600022PubMedID: 16579600Scopus ID: 2-s2.0-33645660631OAI: oai:DiVA.org:kth-5012DiVA: diva2:7466
Note

Uppdaterad från manuskript till artikel: 20101008. QC 20101008

Available 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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Stemme, Göran

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