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Methods for the survey and genetic analysis of populations

  • US 6,613,520 B2
  • Filed: 04/10/2001
  • Issued: 09/02/2003
  • Est. Priority Date: 04/10/2000
  • Status: Expired due to Term
First Claim
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1. A method to determine the genetic diversity of a population by determining the DNA sequence of a targeted polymorphic region from one gene that is common to all, or the majority of all, members of the population, comprising the steps of:

  • a) providing a sample containing a population that comprises at least one genomic DNA molecule containing regions of high sequence conservation flanking a region of polymorphic sequence;

    b) isolating the genomic DNA molecule from the sample;

    c) performing PCR on the genomic DNA molecule, to amplify a subregion of the region of polymorphic sequence, using an upstream primer comprising a binding site for a solid support and a downstream primer to produce an amplified fragment, wherein the upstream primer and the downstream primer hybridize to the regions of high sequence conservation flanking the region of polymorphic sequence;

    d) cleaving the amplified fragment with a restriction enzyme that cleaves the amplified fragment at a site adjacent to the region of polymorphic sequence;

    e) binding the cleaved amplified fragment to a solid support to form an immobilized product;

    f) splitting the immobilized product into two pools;

    g) attaching a double-stranded linker to the immobilized product of each pool, wherein the linker comprises a Type IIS restriction enzyme site;

    h) digesting the immobilized product with a Type IIS restriction enzyme to form a tag;

    i) ligating the tag to a second tag to form a ditag;

    j) amplifying the ditag by PCR;

    k) cleaving the ditag with the restriction enzyme of step d) and ligating the ditag to a second ditag to form a concatemer; and

    l) determining a DNA sequence of the concatemer, wherein the sequence of the concatemer is a sequence of a multitude of markers, which provides a measure of the genetic diversity of a population, thereby allowing for the determination of the genetic diversity of a population.

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