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Bridge DNA amplification of cancer-associated genes on cross-linked agarose microbeads

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Abstract

A cross-linked agarose substrate was studied as a 3D support for bridge solid-phase DNA amplification (SPA). In this kind of SPA, primers are immobilized on agarose beads. Flow cell studies of SPA in real-time experiments showed that the amplification efficiency is strongly affected by (a) the presence of a linker between the primer and substrate, and (b) by the loading with primers. In fact, a high loading density may compromise SPA. The analysis of real time SPA curves using geometric growth model highlighted the advantage of 3D agarose support over the flat surfaces. The potential of bridge 3D SPA in DNA diagnostics was successfully demonstrated by on-chip analysis of mutations of the cancer-associated genes BRCA1/2 and CHEK2.

A cross-linked agarose substrate was studied as a 3D support for bridge solid-phase DNA amplification (SPA). In this kind of SPA, primers are immobilized on agarose beads. The potential of bridge 3D SPA in DNA diagnostics was successfully demonstrated by on-chip analysis of mutations of the cancer-associated genes BRCA1/2 and CHEK2.

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Authors and Affiliations

  1. W. A. Engelhardt Institute of Molecular Biology Russian Academy of Sciences, 119991, Moscow, Russian Federation

    Alexander V. Chudinov, Natalia A. Kolganova, Anton E. Egorov, Denis O. Fesenko, Viktoria E. Kuznetsova, Tatiana V. Nasedkina, Vadim A. Vasiliskov, Alexander S. Zasedatelev & Edward N. Timofeev

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  1. Alexander V. Chudinov
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  2. Natalia A. Kolganova
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  3. Anton E. Egorov
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  4. Denis O. Fesenko
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  5. Viktoria E. Kuznetsova
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  6. Tatiana V. Nasedkina
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  7. Vadim A. Vasiliskov
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  8. Alexander S. Zasedatelev
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  9. Edward N. Timofeev
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Correspondence to Edward N. Timofeev.

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Chudinov, A.V., Kolganova, N.A., Egorov, A.E. et al. Bridge DNA amplification of cancer-associated genes on cross-linked agarose microbeads. Microchim Acta 182, 557–563 (2015). https://doi.org/10.1007/s00604-014-1357-8

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  • DOI: https://doi.org/10.1007/s00604-014-1357-8

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