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Restriction enzyme genotyping

  • US 7,435,541 B2
  • Filed: 04/04/2002
  • Issued: 10/14/2008
  • 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, 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 having the selected polymorphic site within an overhanging end;

    (c) treating the nucleic acid fragment generated in step (b) to convert the overhanging end to a blunt end by extension of the recessed portion of the overhanging end; and

    (d) analyzing one or both strands of the nucleic acid fragment generated in step (c) to identify the nucleotide present at the selected polymorphic site.

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