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Iterative and regenerative DNA sequencing method

  • US 6,258,533 B1
  • Filed: 03/05/1998
  • Issued: 07/10/2001
  • Est. Priority Date: 11/01/1996
  • Status: Expired due to Fees
First Claim
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1. A sequencing method for identifying a first nucleotide n and a second nucleotide n+x in a double stranded nucleic acid segment, comprising:

  • a) digesting said double stranded nucleic acid segment with a restriction enzyme whose cleavage site is separate from its recognition site to produce a double stranded molecule having a single stranded overhang sequence corresponding to an enzyme cut site;

    b) providing an adaptor having a cycle identification tag, a restriction enzyme recognition domain and a sequence identification region;

    c) hybridizing said adaptor to said double stranded nucleic acid having said single-stranded overhang sequence to form a ligated molecule;

    d) amplifying said ligated molecule from step (c) with a labeled primer specific for said cycle identification tag, restriction enzyme recognition domain, and a portion of said sequence identification region of said adaptor;

    e) identifying said nucleotide n by identifying said primer incorporated into the amplification product; and

    f) repeating steps (a) through (e) on said amplified molecule from step (e) to yield the identity of said nucleotide n+x, wherein x is less than or equal to the number of nucleotides between a recognition domain for a restriction enzyme and an enzyme cut site.

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