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Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

  • US 6,780,591 B2
  • Filed: 08/28/2001
  • Issued: 08/24/2004
  • Est. Priority Date: 05/01/1998
  • Status: Active Grant
First Claim
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1. A method of DNA sequencing comprising the steps of:

  • (a) providing a template system comprising at least one nucleic acid molecule of unknown sequence hybridized to a primer oligonucleotide in the presence of a DNA polymerase with reduce exonuclease activity;

    (b) contacting the template system with a single type of deoxyribonucleotide under conditions which allow extension of the primer by incorporation of at least one deoxyribonucleotide having a fluorescent moiety to the 3′

    end of the primer to form an extended primer;

    (c) detecting whether extension of the primer has occurred by detecting a fluorescent signal emitted by the fluorescent moiety, and further comprising destroying the fluorescent signal without removal of the fluorescent moiety;

    (d) detecting the number of deoxyribonucleotides incorporated into the primer;

    (e) removing unincorporated deoxyribonucleotide; and

    (f) repeating steps (a) through (e) to determine the nucleotide sequence of the nucleic acid molecule.

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