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Compositions and methods for nucleotide sequencing

  • US 8,399,188 B2
  • Filed: 09/04/2007
  • Issued: 03/19/2013
  • Est. Priority Date: 09/28/2006
  • Status: Active Grant
First Claim
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1. A method for sequencing a nucleic acid molecule, comprising:

  • (a) contacting a nucleic acid template, a nucleic acid primer complementary to a portion of said template, an extension enzyme, and a reversibly blocked nucleotide under conditions wherein said primer is extended to incorporate said nucleotide into a sequence complementary to said nucleic acid template,said reversibly blocked nucleotide comprising a sugar moiety and a base moiety, wherein said base moiety is linked to a label by an enzymatically cleavable peptide linker, andwherein, attached at the 2′

    or 4′

    position of said sugar moiety, is a reversible blocking group comprising —

    CH2

    CH═

    CH2, —

    CH2

    N3, —

    CH2

    S—

    S-tBu, —

    CH2

    S-Me, —

    CH2

    O—

    CH2

    CH2

    CN, —

    CH2

    CH2

    CN, —

    C(O)—

    CH3, —

    CH3, 2-nitrobenzyl, tetrandyropyran, 4-nitrobenzoyl, 2-aminobenzoyl, —

    C(O)-linker-dye, 2-nitrobenzyl-linker-dye, —

    C(O)—

    O—

    CH2

    CH═

    CH-linker-dye, —

    NH—

    C(O)—

    CH3, —

    F, —

    NH2, phosphate, or sulfate,(b) identifying the nucleotide in said sequence complementary to said nucleic acid template;

    (c1) enzymatically cleaving the peptide linker; and

    (c2) enzymatically removing the blocking group,wherein identifying said nucleotide identifies at least a portion of the sequence of said nucleic acid molecule.

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