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Mutational scanning of PCR products by subtractive oligonucleotide hybridization analysis
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0002-4657-8532
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-4313-1601
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0001-8993-048X
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1999 (English)In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 26, no 2, 308-+ p.Article in journal (Refereed) Published
Abstract [en]

Here, we describe a new approach for mutational scanning of PCR products through hybridization analysis between complementary oligonucleotides. Sets of overlapping probe oligonucleotides complementary to wild-type (WT) sequence are hybridized to microbead-immobilized PCR products under solution-like conditions. Mismatch-hybridization situations between a mutant sample and probe oligonucleotides result in higher remaining concentrations in solution of involved probe oligonucleotides. Post-hybridization supernatants are subsequently analyzed for their probe oligonucleotide compositions using surface plasmon resonance-based biosensor technology Relative remaining probe oligonucleotide concentrations are monitored in real-time through hybridization analysis between probe oligonucleotides and their corresponding sensor-chip immobilized complementary counterparts. This allows for the construction of composition diagrams revealing the existence and approximate location of a mutation within an investigated sample DNA sequence. Applied on PCR products derived from clinical samples of microdissected tumor biopsies, single mutations in exons 6 and 7 of the human p53 tumor-suppressor gene were successfully detected and approximately localized.

Place, publisher, year, edition, pages
1999. Vol. 26, no 2, 308-+ p.
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-13318ISI: 000078631700024OAI: oai:DiVA.org:kth-13318DiVA: diva2:323696
Note
QC 20100611Available from: 2010-06-11 Created: 2010-06-11 Last updated: 2017-12-12Bibliographically 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

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Uhlen, MathiasNygren, Per-Åke

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