| 1. |
- Nilsson, Peter, et al.
(författare)
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Detection of mutations in PCR products from clinical samples by surface plasmon resonance
- 1997
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Ingår i: Journal of Molecular Recognition. - 0952-3499 .- 1099-1352. ; 10:1, s. 7-17
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Nilsson, Peter, et al.
(författare)
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Mutational scanning of PCR products by subtractive oligonucleotide hybridization analysis
- 1999
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Ingår i: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 26:2, s. 308-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- NILSSON, PETER, et al.
(författare)
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REAL-TIME MONITORING OF DNA MANIPULATIONS USING BIOSENSOR TECHNOLOGY
- 1995
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 224:1, s. 400-408
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- O'Meara, Deirdre, et al.
(författare)
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Capture of single-stranded DNA assisted by oligonucleotide modules
- 1998
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 255:2, s. 195-203
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Persson, Björn, et al.
(författare)
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Analysis of oligonucleotide probe affinities using surface plasmon resonance : A means for mutational scanning
- 1997
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 246:1, s. 34-44
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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