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Genotyping by mass spectrometric analysis of allelic fragments

  • US 6,777,188 B2
  • Filed: 03/26/2002
  • Issued: 08/17/2004
  • Est. Priority Date: 10/01/1998
  • Status: Expired due to Fees
First Claim
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1. A method for genotyping a diploid organism, comprising:

  • providing two alleles of a target gene of a diploid organism suspected to contain a polymorphism;

    obtaining a first segment of one allele wherein the segment contains the suspected polymorphic locus;

    obtaining a second segment from the other allele wherein the segment also contains the suspected polymorphic locus;

    replacing a natural nucleotide at greater than 90% of its points of occurrence in the first and the second segment with a modified nucleotide to give a first and a second modified segment;

    wherein, the natural nucleotide that is replaced is not a nucleotide involved in the polymorphism;

    replacing the natural nucleotide with a modified nucleotide comprises amplification using a primer that hybridizes to each segment such that, after amplification, a first modified nucleotide is incorporated between the 3′

    end of the primer and the polymorphic locus; and

    , a second modified nucleotide is located from 5 to 20 nucleotides downstream of the first modified nucleotide;

    cleaving the first and second modified segments at greater than 90% of the points of occurrence of the modified nucleotide to give a first and second set of fragments each of which comprises a 5-20 nucleotide fragment; and

    , comparing masses of the 5-20 nucleotide fragments obtained from the first and the second modified segment to detect the presence or absence of the polymorphism.

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