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
Analysis of oligonucleotide probe affinities using surface plasmon resonance: A means for mutational scanning
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0002-4657-8532
Show others and affiliations
1997 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 246, no 1, 34-44 p.Article in journal (Refereed) Published
Abstract [en]

A novel strategy for real-time analysis of oligonucleotide probe hybridization based on detection by surface plasmon resonance is described. The design of the analysis, exploiting the rapid dissociation kinetics of short oligonucleotides from their hybridization templates, allows monitoring in genuine sensor mode of equilibrium hybridization responses, circumventing the need for regeneration between sample cycles. Applied to a model system comprising oligonucleotide probes and different immobilized hybridization targets the effects of temperature, probe length, and nucleotide substitutions in template were investigated. The procedure described was observed to have an efficient discriminatory power with respect to end-mismatch situations. Affinity determinations of octamer probes showed good correlation between calculated T-m-values and probe affinities. From affinity data collected at different temperatures thermodynamic parameters were determined, which correlated well with data obtained from theoretical calculations. The technique, modified to a simplified form, allowed detection of single nucleotide substitutions in a target template, suggesting that procedures for confirmatory DNA sequencing can be envisioned.

Place, publisher, year, edition, pages
1997. Vol. 246, no 1, 34-44 p.
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-13321ISI: A1997WL81800006OAI: oai:DiVA.org:kth-13321DiVA: diva2:323615
Note
QC 20100611Available from: 2010-06-11 Created: 2010-06-11 Last updated: 2010-06-11Bibliographically approved
In thesis
1. Biosensor technology applied to hybridization analysis and mutation detection
Open this publication in new window or tab >>Biosensor technology applied to hybridization analysis and mutation detection
1998 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

This thesis demonstrates the application of biosensor technology for molecular biology investigations, utilizing a surface plasmon resonance based optical device for mass sensitive detection of biomolecular interactions at a chipsurface. Oligonucleotide model systems were designed for analysis of the action of DNA manipulating enzymes. DNA ligation, DNA cleavage and DNA synthesis could be quantitatively monitored in real-time. A protocol for DNA minisequencing was also established based on prevention of chain elongation by incorporation of chain-terminators. Determinations of affinities for short oligonucleotides hybridizing to an immobilized target were performed with various sequence content, length, temperature and degree of complementarity. The decrease in affinity for hybridizations involving mismatch situations was found to be strongly dependent on the relative position of the mismatch. Interestingly, also end-mismatches were clearly detectable. The stabilization effect achieved upon co-hybridization of two adjacently annealing short oligonucleotide modules (modular primer effect) was also investigated for different module combinations and hybridization situations. The modular concept of hybridizations was subsequently demonstrated to result in enhanced Capture of single stranded PCR products. The sequence based DNA analysis, first introduced with oligonucleotide modelsystems, was extended to the scanning and screening formutations in PCR amplified DNA from clinically relevant samples. Several different formats were investigated, eitherwith the PCR products immobilized on the chip and oligonucleotides injected or vice versa. Again, mismatch discrimination could be observed for wild type and mutant specific oligonucleotides hybridizing to the targets. The experimental set-up for mutation detection was further developed by the introduction of a subtractive mismatch sensitive hybridization outside the instrument and a subsequent determination of the relative amounts of remain ingoligonucleotides with analytical biosensor monitoring of hybridizations between fully complementary oligonucleotides. In conclusion, the applied technology was found to be a suitable tool for a wide range of molecular biology applications, with emphasis on hybridization analysis and mutation detection.

Place, publisher, year, edition, pages
Stockholm: KTH, 1998. 56 p.
Keyword
biosensor, genosensor, surface plasmon resonance, hybridization, nucleic acids, oligonucleotide, mutation detection, mismatch discrimination, modular
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-2703 (URN)91-7170-322-5 (ISBN)
Public defence
1998-11-06, 00:00
Note
QC 20100611Available from: 2000-01-01 Created: 2000-01-01 Last updated: 2010-06-11Bibliographically approved

Open Access in DiVA

No full text

Authority records BETA

Uhlen, MathiasNygren, Per-Åke

Search in DiVA

By author/editor
Nilsson, PeterUhlen, MathiasNygren, Per-Åke
By organisation
Biotechnology
In the same journal
Analytical Biochemistry
Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 35 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