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Multiplex genotyping using solid phase capturable dideoxynucleotides and mass spectrometry

  • US 20060252038A1
  • Filed: 07/11/2003
  • Published: 11/09/2006
  • Est. Priority Date: 07/12/2002
  • Status: Abandoned Application
First Claim
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1. A method for determining the identity of a nucleotide present at a predetermined site in a DNA whose sequence immediately 3′

  • of such predetermined site is known which comprises;

    (a) treating the DNA with an oligonucleotide primer whose sequence is complementary to such known sequence so that the oligonucleotide primer hybridizes to the DNA and forms a complex in which the 3′

    end of the oligonucleotide primer is located immediately adjacent to the predetermined site in the DNA;

    (b) simultaneously contacting the complex from step (a) with four different labeled dideoxynucleotides, in the presence of a polymerase under conditions permitting a labeled dideoxynucleotide to be added to the 3′

    end of the primer so as to generate a labeled single base extended primer, wherein each of the four different labeled dideoxynucleotides (i) is complementary to one of the four nucleotides present in the DNA and (ii) has a molecular weight which can be distinguished from the molecular weight of the other three labeled dideoxynucleotides using mass spectrometry; and

    (c) determining the difference in molecular weight between the labeled single base extended primer and the oligonucleotide primer so as to identify the dideoxynucleotide incorporated into the single base extended primer and thereby determine the identity of the nucleotide present at the predetermined site in the DNA.

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