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Methods for genetic analysis of DNA to detect sequence variances

  • US 8,129,120 B2
  • Filed: 02/28/2007
  • Issued: 03/06/2012
  • Est. Priority Date: 05/23/2000
  • Status: Expired due to Fees
First Claim
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1. A method for determining the nucleotide present at a selected polymorphic site in a target nucleic acid molecule, the method comprising:

  • a) amplifying a portion of the target nucleic acid molecule comprising the selected polymorphic site using a first primer and a second primer, the second primer containing a recognition site for a first restriction enzyme and a recognition site for a second restriction enzyme, to generate amplification product containing a recognition site for the first restriction enzyme and a recognition site for the second restriction enzyme such that digestion of the amplification product with the first restriction enzyme and the second restriction enzyme generates a nucleic acid fragment containing the selected polymorphic site;

    b) digesting the amplification product with the first restriction enzyme and the second restriction enzyme to generate a nucleic acid fragment containing the selected polymorphic site within a 5′

    single-stranded overhang;

    c) filling the recessed end corresponding to the 5′

    overhang with nucleotides wherein at least one of the nucleotides is bromodeoxuridine; and

    d) analyzing the nucleic acid fragment to identify the nucleotide present at the selected polymorphic site.

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