Patent ID: 6613520
Filing Date: 2003-09-02
Classification: C12Q,G16B,Y10T

Abstract:
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.