×

DNA extension and analysis with rolling primers

  • US 5,962,228 A
  • Filed: 08/22/1997
  • Issued: 10/05/1999
  • Est. Priority Date: 11/17/1995
  • Status: Expired due to Fees
First Claim
Patent Images

1. A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of:

  • (a) providing a set of first primers, each first primer of the set having a 3'"'"'-terminal nucleotide, a template positioning segment, and an extension region comprising one or more complexity-reducing nucleotides;

    (b) providing a double stranded DNA template comprising a first primer binding site, a promoter, the polynucleotide, and a second primer binding site, the first primer binding site being capable of forming an extendable duplex with at least one of the first primers;

    (c) generating a population of RNA transcripts from the double stranded DNA template with an RNA polymerase that recognizes the promoter;

    (d) mutating the first primer binding site in the RNA transcripts by extending a first primer forming an extendable duplex therewith, so that the first primer binding site is shifted one nucleotide in the direction of extension and so that a single stranded DNA template is formed;

    (e) forming an amplicon from the single stranded DNA template;

    (f) identifying the 3'"'"'-terminal nucleotide of the first primer extended to form the single stranded DNA template by the identity of the amplicon; and

    (g) repeating steps (b) through (f) until the nucleotide sequence of the polynucleotide is determined.

View all claims
  • 2 Assignments
Timeline View
Assignment View
    ×
    ×