Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Microfluidic bead-based methods for DNA analysis
KTH, School of Electrical Engineering (EES).
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 [en]
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
Keyword [sv]
Genetik
National Category
Medical Genetics
Identifiers
URN: urn:nbn:se:kth:diva-155ISBN: 91-7283-992-9 (print)OAI: oai:DiVA.org:kth-155DiVA: diva2:7467
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
List of papers
1. SNP analysis by allele-specific extension in a micromachined filter chamber
Open this publication in new window or tab >>SNP analysis by allele-specific extension in a micromachined filter chamber
Show others...
2002 (English)In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 32, no 4, 748-754 p.Article in journal (Refereed) Published
Keyword
SINGLE-NUCLEOTIDE POLYMORPHISMS, HUMAN GENOME, HYBRIDIZATION, SYSTEM, PROBES, DNA
National Category
Medical Genetics
Identifiers
urn:nbn:se:kth:diva-5007 (URN)000174881200006 ()11962595 (PubMedID)
Note
QC 20100929 NR 20140805Available from: 2005-04-01 Created: 2005-04-01 Last updated: 2017-12-05Bibliographically approved
2. Single-nucleotide polymorphism analysis by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device
Open this publication in new window or tab >>Single-nucleotide polymorphism analysis by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device
Show others...
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.

Keyword
filter chamber, flow-through, fluorescence, microfluidics, single-nucleotide polymorphism
National Category
Medical Genetics
Identifiers
urn:nbn:se:kth:diva-5008 (URN)10.1002/elps.200390008 (DOI)000180637300021 ()12652586 (PubMedID)
Note
QC 20100929 NR 20140805Available from: 2005-04-01 Created: 2005-04-01 Last updated: 2017-12-05Bibliographically approved
3. Single nucleotide polymorphism analysis by allele-specific primer extension with real-time bioluminescence detection in a microfluidic device
Open this publication in new window or tab >>Single nucleotide polymorphism analysis by allele-specific primer extension with real-time bioluminescence detection in a microfluidic device
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.

Keyword
Microfluidics, Nucleotide polymorphism, Nucleotides, Primer extension, Pyrosequencing
National Category
Medical Genetics
Identifiers
urn:nbn:se:kth:diva-5009 (URN)10.1016/S0021-9673(03)01033-1 (DOI)000185557200005 ()
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
4. Pyrosequencing in a microfluidic flow-through device
Open this publication in new window or tab >>Pyrosequencing in a microfluidic flow-through device
2005 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 77, no 23, 7505-7511 p.Article in journal (Refereed) Published
Abstract [en]

To explore genome variation meaningfully, there is a critical need for a high-throughput and inexpensive platform for DNA analysis. Pyrosequencing is a nonelectrophoretic bioluminometric DNA sequencing method that uses a four-enzyme mixture reaction to monitor nucleotide incorporation in real time. Currently, the commercialized pyrosequencing technique is limited to a 96-microtiter plate format. However, high throughput and inexpensive pyrosequencing is required to meet the need of screening large numbers of samples. We present here DNA pyrosequencing on a nanoliter-volume microfluidic platform. The microfluidic approach involves the trapping of the DNA on microbeads in an on-chip filter chamber and flow-through of the pyrosequencing reagents to monitor the reaction in real time. Two single-nucleotide polymorphisms were successfully scored to evaluate the microfluidic platform. In addition to significantly reducing reagent costs, microfluidic systems promise to improve the read length by eliminating intermediate product accumulation by constant removal of unincorporated nucleotides and elimination of dilution effects at each reaction cycle in the current plate format. Although only one filter chamber was used in this study, the platform should be readily adaptable to parallel analyses of nanoliter samples using filter chamber arrays to obtain high-throughput DNA analysis.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2005
Keyword
single-nucleotide polymorphisms, real-time, filter-chamber, dna, analysis, beads, discrimination, hybridization, extension, probes
National Category
Medical Genetics
Identifiers
urn:nbn:se:kth:diva-15238 (URN)10.1021/ac0507542 (DOI)000233785900012 ()16316155 (PubMedID)2-s2.0-28544436911 (Scopus ID)
Note

QC 20100525

Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
5. Genotyping by dynamic heating of monolayered beads on a microheated surface
Open this publication in new window or tab >>Genotyping by dynamic heating of monolayered beads on a microheated surface
Show others...
2004 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 25, no 21-22, 3712-3719 p.Article in journal (Refereed) Published
Abstract [en]

 A miniaturized bead-based dynamic allele-specific hybridization (DASH) approach for sing le-nucleotide polymorphism analysis is presented. Chips with integrated heater and temperature sensors for open-surface DNA analysis were microfabricated. Microcontact printing using a poly(dimethylsiloxane) (PDMS) stamp was employed to create monolayers of immobilized beads on the surface of the chip. This chip allows fast, well-controllable temperature ramping. The temperature distribution was homogeneous over the entire heater area. All three possible variants of an SNP site of a synthesized oligonucleotide were accurately scored using the bead-based DASH approach. Our assay has a nonoptimized temperature ramping rate of 4degreesC-6degreesC/min compared to earlier reported values of 2degreesC-3degreesC/min, thereby reducing the total analysis time by a factor of 2. Reliable DASH measurement data from areas as small as 12 x 13 mum was achieved. Our bead-based DASH approach has enabled a dramatic volume reduction and is a step towards developing a cost-effective high-throughput DASH method on arrays of single beads.

Keyword
beads, dynamic allele-specific hybridization, microcontact printing, microheater, miniaturization, single-nucleotide polymorphism
National Category
Medical Genetics
Identifiers
urn:nbn:se:kth:diva-5011 (URN)10.1002/elps.200406065 (DOI)000225679000021 ()2-s2.0-10944219819 (Scopus ID)
Note

QC 20100916 20110915

Available from: 2005-04-01 Created: 2005-04-01 Last updated: 2017-04-26Bibliographically approved
6. Rapid melting curve analysis on monolayered beads for high-throughput genotyping of single-nucleotide polymorphisms
Open this publication in new window or tab >>Rapid melting curve analysis on monolayered beads for high-throughput genotyping of single-nucleotide polymorphisms
Show others...
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
Keyword
allele-specific hybridization, real-time, dna, system, arrays
National Category
Medical Genetics
Identifiers
urn:nbn:se:kth:diva-5012 (URN)10.1021/ac051771u (DOI)000236686600022 ()16579600 (PubMedID)2-s2.0-33645660631 (Scopus ID)
Note

Uppdaterad från manuskript till artikel: 20101008. QC 20101008

Available from: 2005-04-01 Created: 2005-04-01 Last updated: 2017-12-05Bibliographically approved

Open Access in DiVA

fulltext(3593 kB)3530 downloads
File information
File name FULLTEXT01.pdfFile size 3593 kBChecksum MD5
c17700ed50f1b0fea99ceb66286e8617296ae46d70f3650d7a7f91a20f433698333ded68
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Russom, Aman
By organisation
School of Electrical Engineering (EES)
Medical Genetics

Search outside of DiVA

GoogleGoogle Scholar
Total: 3530 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 1343 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf