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Isothermal methods for creating clonal single molecule arrays

  • US 20080009420A1
  • Filed: 03/19/2007
  • Published: 01/10/2008
  • Est. Priority Date: 03/17/2006
  • Status: Abandoned Application
First Claim
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1. A method for isothermally amplifying single stranded nucleic acid molecules immobilized on a planar solid surface comprising:

  • i) providing a planar solid surface comprising at least one 5′

    -end immobilized first single stranded nucleic acid template molecule comprising a sequence Y at the 5′

    end and a sequence Z at the 3′

    end and a plurality of first and second primers comprising sequences X and Y immobilized at their 5′

    ends, wherein sequence X is hybridizable to sequence Z;

    ii) annealing said at least one 5′

    -end immobilized first single stranded nucleic acid template molecule to said first immobilized primers, wherein the first sequence Z of each template molecule is annealed to one of said first immobilized primers comprising sequence X;

    iii) performing a primer extension reaction using primer annealed 5′

    -end immobilized first single stranded nucleic acid template molecules to generate double stranded nucleic acid molecules comprising 5′

    -end immobilized first and second single stranded nucleic acid molecules, wherein the 5′

    -end immobilized second single stranded nucleic acid molecules are complementary copies of the 5′

    -end immobilized first single stranded template nucleic acid molecules and each of the 5′

    -end immobilized second single stranded nucleic acid molecules comprises a sequence at the 3′

    end that is hybridizable to the second primer sequence Y;

    iv) flowing a chemical denaturant across the planar solid surface to denature said double stranded nucleic acid molecules to generate 5′

    -end immobilized first and second single stranded nucleic acid molecules;

    v) removing the chemical denaturant and annealing said 5′

    -end immobilized first and second single stranded nucleic acid molecules to said first and second immobilized primers comprising sequences X and Y;

    vi) performing a primer extension reaction using primer annealed 5′

    -end immobilized first and second single stranded nucleic acid molecules as templates to generate double stranded nucleic acid molecules immobilized at both 5′

    -ends; and

    vii) repeating steps iv) through vi) to generate multiple copies of the nucleic acid molecules on said planar solid surface, wherein steps iv) through vi) are carried out at the same temperature.

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