Rapid Direct Sequence Analysis of Disease Genes by Use of Nested Primers
The present invention provides a method that allows direct sequence analysis of disease genes, in a rapid, accurate, and economical fashion. This method, single condition amplification/internal primer (SCAIP) sequencing, relies on amplification of a large number of exons at a single set of PCR temperatures. Sequencing specificity is achieved by uniform use of a second, nested internal set of sequencing primers. The benefits of this technique are especially apparent in analysis of sequence of large multi-exon genes, including the dystrophin gene � the largest human gene yet known. Mutations in this large gene are responsible for the common and devastating disorder Duchenne Muscular Dystrophy (DMD). The inventors have demonstrated the practicability of this method in patients affected by mutations in dystrophin gene.
Direct sequence analysis for mutations in the genes responsible for many human diseases has not been economically feasible, mainly because of the technical difficulties inherent in obtaining high-quality and reliable sequencing data. This invention allows rapid, sensitive, reliable, and economical gene mutation analysis for confirmation of diagnosis, or determination of disease carrier state, in many human diseases. For some of these diseases, determination of the specific mutation may determine the exact nature of therapy. For example, patients with premature stop codon mutations as determined by this assay may be candidates for therapies that have the potential of responding to readthrough of premature stop codons.
Stage of Development
A formal patent application has been filed with the U.S. Patent and Trademark Office (US #20060223062). Nationalized PCT applications are pending in Canada, Europe and Australia.
This technology is available for licensing under either exclusive or non-exclusive terms.
*lanigan KM, von Niederhausern A, Dunn DM, Alder J, Mendell JR, Weiss RB. (2003) Rapid direct sequence analysis of the dystrophin gene. Am J Hum Genet. 72(4):931-9.
*Dent KM, Dunn DM, von Niederhausern AC, Aoyagi AT, Kerr L, Bromberg MB, Hart KJ, Tuohy T, White S, den Dunnen JT, Weiss RB, Flanigan KM. (2005) Improved molecular diagnosis of dystrophinopathies in an unselected clinical cohort. Am J Med Genet A. 134(3):295-8.
Kevin Flanigan, Robert Weiss, Andrew Von Niederhausen, Diane Dunn
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