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Automatable process for sequencing nucleotide

  • US 4,863,849 A
  • Filed: 07/18/1985
  • Issued: 09/05/1989
  • Est. Priority Date: 07/18/1985
  • Status: Expired due to Term
First Claim
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1. A method for determining the base sequence of nucleotides comprising:

  • (a) adding an activated nucleoside 5'"'"'-triphosphate precursor of one known nitrogenous base composition to a reaction mixture comprising a template-directed nucleotide polymerase and a single-stranded polynucleotide templates hybridized to complementary oligonucleotide primer strands at least one nucleotide residue shorter than the templates to form at least one unpaired nucleotide residue in each template at the 3'"'"' -end of the primer strand under reaction conditions which allow incorporation of the activated nucleoside 5'"'"'-triphosphate precursor onto the 3'"'"'-end of the primer strands, provided the nitrogenous base of the activated nucleoside 5'"'"'-triphosphate precursor is complementary to the nitrogenous base of the unpaired nucleotide residue of the templates;

    (b) detecting whether or not the nucleoside 5'"'"'triphosphate precursor was incorporated into the primer strands in which incorporation of the nucleoside 5'"'"'-triphosphate precursor indicates that the unpaired nucleotide residue of the template has a nitrogenous base composition that is complementary to that of the incorporated nucleoside 5'"'"'-triposphate precursor; and

    (c) sequentially repeating steps (a) and (b), wherein each sequential repetition adds and, detects the incorporation of one type of activated nucleoside 5'"'"'-triphosphate precursor of known nitrogenous base composition; and

    (d) determining the base sequence of the unpaired nucleotide residues of the template from the sequence of incorporation of said nucleoside precursors.

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