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Biosensor technology applied to hybridization analysis and mutation detection
KTH, Superseded Departments, Biotechnology.
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 [en]
biosensor, genosensor, surface plasmon resonance, hybridization, nucleic acids, oligonucleotide, mutation detection, mismatch discrimination, modular
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-2703ISBN: 91-7170-322-5 (print)OAI: oai:DiVA.org:kth-2703DiVA: diva2:8406
Public defence
1998-11-06, 00:00
Note
QC 20100611Available from: 2000-01-01 Created: 2000-01-01 Last updated: 2010-06-11Bibliographically approved
List of papers
1. REAL-TIME MONITORING OF DNA MANIPULATIONS USING BIOSENSOR TECHNOLOGY
Open this publication in new window or tab >>REAL-TIME MONITORING OF DNA MANIPULATIONS USING BIOSENSOR TECHNOLOGY
1995 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 224, no 1, 400-408 p.Article in journal (Refereed) Published
Abstract [en]

The potential of real-time biospecific interaction analysis technology for applications in molecular biology is described. DNA fragments are immobilized onto a biosensor surface using the high-affinity streptavidin-biotin system and subsequently used to monitor different unit operations in molecular biology, e.g., DNA strand separation, DNA hybridization kinetics, and enzymatic modifications. A model system comprising six oligonucleotides was used, which can be assembled into a 69-bp double-stranded DNA fragment. Using this system, the biosensor approach was employed to analyze multistep solid-phase gene assembly and the performance of different enzymes routinely used for the synthesis and manipulation of DNA. In addition, a concept for the determination of single-point mutations in DNA samples is described.

National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-13317 (URN)A1995QB30600057 ()
Note

QC 20100611

Available from: 2010-06-11 Created: 2010-06-11 Last updated: 2017-12-12Bibliographically approved
2. Analysis of oligonucleotide probe affinities using surface plasmon resonance: A means for mutational scanning
Open this publication in new window or tab >>Analysis of oligonucleotide probe affinities using surface plasmon resonance: A means for mutational scanning
Show others...
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.

National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-13321 (URN)A1997WL81800006 ()
Note
QC 20100611Available from: 2010-06-11 Created: 2010-06-11 Last updated: 2017-12-12Bibliographically approved
3. Quantitative Investigation of the Modular Primer Effect for DNA and Peptide Nucleic Acid Hexamers
Open this publication in new window or tab >>Quantitative Investigation of the Modular Primer Effect for DNA and Peptide Nucleic Acid Hexamers
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1999 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 269, no 1, 155-161 p.Article in journal (Refereed) Published
Abstract [en]

The effect on oligonucleotide–template duplex stability upon cohybridization of adjacently annealing oligonucleotides, the modular primer effect, was studied with biosensor technology. DNA and peptide nucleic acid (PNA) hexamer modules and sensor chip-immobilized template DNA strands were designed for analysis of nick, overlap, and gap modular hybridization situations. The fast hybridization kinetics for such hexamer modules allowed for the determination of apparent duplex affinities from equilibrium responses. The results showed that the hybridizational stability of modular hexamer pairs is strongly dependent on the positioning, concentration, and inherent affinity of the adjacently annealing hexamer module. Up to 80-fold increases in apparent affinities could be observed for adjacent modular oligonucleotide pairs compared to affinities determined for single hexamer oligonucleotide hybridizations. Interestingly, also for coinjections of different module combinations where DNA hexamer modules were replaced by their PNA counterparts, a modular primer effect was observed. The introduction of a single base gap between two hexamer modules significantly reduced the stabilization effect, whereas a gap of two bases resulted in a complete loss of the effect. The results suggest that the described biosensor-based methodology should be useful for the selection of appropriate modules and working concentrations for use in different modular hybridization applications.

National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-13322 (URN)10.1006/abio.1999.4000 (DOI)
Note
QC 20100611Available from: 2010-06-11 Created: 2010-06-11 Last updated: 2017-12-12Bibliographically approved
4. Capture of single-stranded DNA assisted by oligonucleotide modules
Open this publication in new window or tab >>Capture of single-stranded DNA assisted by oligonucleotide modules
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1998 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 255, no 2, 195-203 p.Article in journal (Refereed) Published
Abstract [en]

Real-time biospecific interaction analysis was employed to monitor direct capture of a hepatitis C virus (HCV) derived polymerase chain reaction (PCR) product by nucleic acid hybridization. Different formats for hybridization were used to study the interaction between a single-stranded HCV PCR product and capture oligonucleotides immobilized on a sensor chip via streptavidin-biotin chemistry. By employing a prehybridization step in solution with nonbiotin oligonucleotides complementary to the single-stranded target and adjacent to the immobilized probe, a significant capture was achieved in comparison to the low capture efficiency obtained using single immobilized probes (9-36 mer). High capture efficiencies were also observed when shorter immobilized probes were used in combination with strings of adjacently positioned prehybridized probes (i.e., modules). Interestingly, the introduction of single nucleotide gaps between prehybridized and/or immobilized probes dramatically reduced the capture efficiency. These results suggest that flexible systems for capture could be designed from libraries of short oligonucleotides (9 mers) used in module fashion, taking advantage of stacking interactions between the oligonucleotides. The potential applications of such oligonucleotide-assisted capture systems are discussed.

National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-13319 (URN)000071557200006 ()
Note
QC 20100611Available from: 2010-06-11 Created: 2010-06-11 Last updated: 2017-12-12Bibliographically approved
5. Detection of mutations in PCR products from clinical samples by surface plasmon resonance
Open this publication in new window or tab >>Detection of mutations in PCR products from clinical samples by surface plasmon resonance
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1997 (English)In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 10, no 1, 7-17 p.Article in journal (Refereed) Published
Abstract [en]

Two different strategies for scanning and screening of mutations in polymerase chain reaction (PCR) products by hybridization analysis are described, employing real-time biospecific interaction analysis (BIA) for detection, Real-time BIA was used to detect differences in hybridization responses between PCR products and different 17-mer oligonucleotide probes, For the analysis using a biosensor instrument, two different experimental formats were investigated based on immobilization of either biotinylated PCR products or oligonucleotide probes onto a sensor chip, Applied on the human tumour suppressor p53 gene, differences in hybridization levels for full-match and mismatch situations employing both formals allowed the detection of point mutations in exon 6 PCR products, derived from a breast tumour biopsy sample, In addition, a mutant sample sequence could be detected in a 50/50 background of wild type exon 6 sequence, The suitability of the different formats for obtaining a regenerable system and a high throughput of samples is discussed.

National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-13320 (URN)A1997XA36200002 ()
Note
QC 20100611Available from: 2010-06-11 Created: 2010-06-11 Last updated: 2017-12-12Bibliographically approved
6. Mutational scanning of PCR products by subtractive oligonucleotide hybridization analysis
Open this publication in new window or tab >>Mutational scanning of PCR products by subtractive oligonucleotide hybridization analysis
Show others...
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.

National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-13318 (URN)000078631700024 ()
Note
QC 20100611Available from: 2010-06-11 Created: 2010-06-11 Last updated: 2017-12-12Bibliographically approved

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