Method for the detection of genetic diseases and gene sequence variations by single nucleotide primer extension

DETAILED DESCRIPTION Among the objects of the invention, therefore, may be noted the provision of a method for the detection of abnormal alleles in those genetic diseases where the frequency of occurrence of the same mutation is high, and in other genetic diseases where multiple mutations cause the disease and the sequence variation in an affected member of a given family is known; the provision of a method for determining the sequence of a genomic DNA sample at a predetermined position thereof; the provision of such a method in which nucleotide sequence variations of as little as one nucleotide can be detected; and the provision of such a method which is relatively rapid and not technically demanding.
Briefly, therefore, the present invention is directed to a method for detecting a known genetic abnormality or gene sequence variation resulting from a nucleotide substitution, translocation, insertion or deletion at a predetermined position in a sample DNA fragment by single nucleotide primer extension.
The method comprises providing the sample DNA fragment as a denatured molecule and mixing it with an inducing agent, a primer having a sequence complementary to a region flanking the predetermined position, and a labeled nucleotide to form a mixture.
The mixture has an essential absence of nucleotides constituted of bases other than the base of which the labeled nucleotide is constituted.
The mixture is subjected to conditions conducive for the annealing of primer to the single stranded molecule and the formation of a primer extension product incorporating the labeled nucleotide.
The mixture is thereafter analyzed for the presence of primer extension product which has incorporated the labeled nucleotide.
Other objects will be in part apparent, and in part pointed out hereinafter .