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  • 1.
    Nilsson, Peter
    et al.
    KTH, Superseded Departments, Biotechnology.
    O'Meara, Deirdre
    KTH, Superseded Departments, Biotechnology.
    Edebratt, Fredrik
    KTH, Superseded Departments, Biotechnology.
    Persson, Björn
    Uhlén, Mattias
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Quantitative Investigation of the Modular Primer Effect for DNA and Peptide Nucleic Acid Hexamers1999In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 269, no 1, p. 155-161Article in journal (Refereed)
    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.

  • 2.
    O'Meara, Deirdre
    et al.
    KTH, Superseded Departments, Biotechnology.
    Nilsson, Peter
    KTH, Superseded Departments, Biotechnology.
    Nygren, Per-Åke
    KTH, Superseded Departments, Biotechnology.
    Uhlen, Mathias
    KTH, Superseded Departments, Biotechnology.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Capture of single-stranded DNA assisted by oligonucleotide modules1998In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 255, no 2, p. 195-203Article in journal (Refereed)
    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.

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