Concentrating and labeling genomic DNA in a nanofluidic array

Rodolphe Marie*, Jonas Nyvold Pedersen, Kalim U. Mir, Brian Bilenberg, Anders Kristensen

*Corresponding author for this work

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    Abstract

    Nucleotide incorporation by DNA polymerase forms the basis of DNA sequencing-by-synthesis. In current platforms, either the single-stranded DNA or the enzyme is immobilized on a solid surface to locate the incorporation of individual nucleotides in space and/or time. Solid-phase reactions may, however, hinder the polymerase activity. We demonstrate a device and a protocol for the enzymatic labeling of genomic DNA arranged in a dense array of single molecules without attaching the enzyme or the DNA to a surface. DNA molecules accumulate in a dense array of pits embedded within a nanoslit due to entropic trapping. We then perform ϕ29 polymerase extension from single-strand nicks created on the trapped molecules to incorporate fluorescent nucleotides into the DNA. The array of entropic traps can be loaded with λ-DNA molecules to more than 90% of capacity at a flow rate of 10 pL min-1. The final concentration can reach up to 100 μg mL-1, and the DNA is eluted from the array by increasing the flow rate. The device may be an important preparative module for carrying out enzymatic processing on DNA extracted from single-cells in a microfluidic chip.
    Original languageEnglish
    JournalNanoscale
    Volume10
    Pages (from-to)1376-1382
    ISSN2040-3364
    DOIs
    Publication statusPublished - 2018

    Cite this

    @article{cfc033dcb1a94b9396770bfe5ce71ad8,
    title = "Concentrating and labeling genomic DNA in a nanofluidic array",
    abstract = "Nucleotide incorporation by DNA polymerase forms the basis of DNA sequencing-by-synthesis. In current platforms, either the single-stranded DNA or the enzyme is immobilized on a solid surface to locate the incorporation of individual nucleotides in space and/or time. Solid-phase reactions may, however, hinder the polymerase activity. We demonstrate a device and a protocol for the enzymatic labeling of genomic DNA arranged in a dense array of single molecules without attaching the enzyme or the DNA to a surface. DNA molecules accumulate in a dense array of pits embedded within a nanoslit due to entropic trapping. We then perform ϕ29 polymerase extension from single-strand nicks created on the trapped molecules to incorporate fluorescent nucleotides into the DNA. The array of entropic traps can be loaded with λ-DNA molecules to more than 90{\%} of capacity at a flow rate of 10 pL min-1. The final concentration can reach up to 100 μg mL-1, and the DNA is eluted from the array by increasing the flow rate. The device may be an important preparative module for carrying out enzymatic processing on DNA extracted from single-cells in a microfluidic chip.",
    author = "Rodolphe Marie and Pedersen, {Jonas Nyvold} and Mir, {Kalim U.} and Brian Bilenberg and Anders Kristensen",
    year = "2018",
    doi = "10.1039/c7nr06016e",
    language = "English",
    volume = "10",
    pages = "1376--1382",
    journal = "Nanoscale",
    issn = "2040-3364",
    publisher = "Royal Society of Chemistry",

    }

    Concentrating and labeling genomic DNA in a nanofluidic array. / Marie, Rodolphe ; Pedersen, Jonas Nyvold; Mir, Kalim U.; Bilenberg, Brian; Kristensen, Anders.

    In: Nanoscale, Vol. 10, 2018, p. 1376-1382 .

    Research output: Contribution to journalJournal articleResearchpeer-review

    TY - JOUR

    T1 - Concentrating and labeling genomic DNA in a nanofluidic array

    AU - Marie, Rodolphe

    AU - Pedersen, Jonas Nyvold

    AU - Mir, Kalim U.

    AU - Bilenberg, Brian

    AU - Kristensen, Anders

    PY - 2018

    Y1 - 2018

    N2 - Nucleotide incorporation by DNA polymerase forms the basis of DNA sequencing-by-synthesis. In current platforms, either the single-stranded DNA or the enzyme is immobilized on a solid surface to locate the incorporation of individual nucleotides in space and/or time. Solid-phase reactions may, however, hinder the polymerase activity. We demonstrate a device and a protocol for the enzymatic labeling of genomic DNA arranged in a dense array of single molecules without attaching the enzyme or the DNA to a surface. DNA molecules accumulate in a dense array of pits embedded within a nanoslit due to entropic trapping. We then perform ϕ29 polymerase extension from single-strand nicks created on the trapped molecules to incorporate fluorescent nucleotides into the DNA. The array of entropic traps can be loaded with λ-DNA molecules to more than 90% of capacity at a flow rate of 10 pL min-1. The final concentration can reach up to 100 μg mL-1, and the DNA is eluted from the array by increasing the flow rate. The device may be an important preparative module for carrying out enzymatic processing on DNA extracted from single-cells in a microfluidic chip.

    AB - Nucleotide incorporation by DNA polymerase forms the basis of DNA sequencing-by-synthesis. In current platforms, either the single-stranded DNA or the enzyme is immobilized on a solid surface to locate the incorporation of individual nucleotides in space and/or time. Solid-phase reactions may, however, hinder the polymerase activity. We demonstrate a device and a protocol for the enzymatic labeling of genomic DNA arranged in a dense array of single molecules without attaching the enzyme or the DNA to a surface. DNA molecules accumulate in a dense array of pits embedded within a nanoslit due to entropic trapping. We then perform ϕ29 polymerase extension from single-strand nicks created on the trapped molecules to incorporate fluorescent nucleotides into the DNA. The array of entropic traps can be loaded with λ-DNA molecules to more than 90% of capacity at a flow rate of 10 pL min-1. The final concentration can reach up to 100 μg mL-1, and the DNA is eluted from the array by increasing the flow rate. The device may be an important preparative module for carrying out enzymatic processing on DNA extracted from single-cells in a microfluidic chip.

    U2 - 10.1039/c7nr06016e

    DO - 10.1039/c7nr06016e

    M3 - Journal article

    VL - 10

    SP - 1376

    EP - 1382

    JO - Nanoscale

    JF - Nanoscale

    SN - 2040-3364

    ER -