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Single nucleotide polymorphism analysis by allele-specific primer extension with real-time bioluminescence detection in a microfluidic device
KTH, Superseded Departments, Signals, Sensors and Systems.
KTH, Superseded Departments, Signals, Sensors and Systems.
KTH, Superseded Departments, Signals, Sensors and Systems.ORCID iD: 0000-0001-9552-4234
2003 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1014, no 1-2, 37-45 p.Article in journal (Refereed) Published
Abstract [en]

A microfluidic approach for rapid bioluminescent real-time detection of single nucleotide polymorphism (SNP) is presented. The method is based on single-step primer extension using pyrosequencing chemistry to monitor nucleotide incorporations in real-time. The method takes advantage of the fact that the reaction kinetics differ between matched and mismatched primer-template configurations. We show here that monitoring the initial reaction in real time accurately scores SNPs by comparing the initial reaction kinetics between matched and mismatched configurations. Thus, no additional treatment is required to improve the sequence specificity of the extension, which has been the case for many allele-specific extension assays. The microfluidic approach was evaluated using four SNPs. Three of the SNPs included primer-template configurations that have been previously reported to be difficult to resolve by allele-specific primer extension. All SNPs investigated were successfully scored. Using the microfluidic device, the volume for the bioluminescent assay was reduced dramatically, thus offering a cost-effective and fast SNP analysis method.

Place, publisher, year, edition, pages
2003. Vol. 1014, no 1-2, 37-45 p.
Keyword [en]
Microfluidics, Nucleotide polymorphism, Nucleotides, Primer extension, Pyrosequencing
National Category
Medical Genetics
Identifiers
URN: urn:nbn:se:kth:diva-5009DOI: 10.1016/S0021-9673(03)01033-1ISI: 000185557200005OAI: oai:DiVA.org:kth-5009DiVA: diva2:7463
Note
16th International Symposium on Microscale Separation and Analysis SAN DIEGO, CALIFORNIA, JAN 17-22, 2003 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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Stemme, Göran

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