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Sequential step method for sequencing and identifying polynucleotides

DC
  • US 5,908,755 A
  • Filed: 10/15/1997
  • Issued: 06/01/1999
  • Est. Priority Date: 06/14/1996
  • Status: Expired due to Term
First Claim
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1. A method of sequential step sequencing of a polynucleotide sequence with two or more iterations of steps (A) through (E) below, wherein the polynucleotide sequence is included on a polynucleotide template, the method comprising:

  • (A) providing the polynucleotide template and a first primer, wherein the first primer has at least three nucleotides and is complementary to a segment of the polynucleotide template;

    (B) causing the template and the first primer to anneal, thereby forming a template-primer complex;

    (C) adding a template-dependent nucleotide polymerase and at least one nucleoside triphosphate or analog thereof having a label attached thereto, wherein each nucleoside triphosphate or analog thereof includes a base selected from the group consisting of adenine, thymine, cytosine, guanine, and uracil;

    (D) detecting a signal from any label incorporated into the template-primer complex;

    (E) if no label is incorporated into the template-primer complex as determined in step (D), then steps (C) and (D) are repeated using another nucleoside triphosphate or analog thereof having a different base than that used previously in step (C), steps (C) and (D) being repeated until it is determined that the label is incorporated into the template-primer complex, thereby identifying which nucleoside triphosphate was added to the 3'"'"' end of the primer; and

    (F) generating a new primer having at least three nucleotides that includes a base or bases identified in the immediately preceding iteration of steps (A) through (E) at the new primer'"'"'s 3'"'"' end, wherein the nucleotide at the 3'"'"' end of the new primer does not have a label attached thereto; and

    steps (A) to (E) are repeated with the proviso that the new primer is substituted for the first primer.

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