Patent ID: 6566054
Filing Date: 2003-05-20
Classification: A01K,C07K,C12N,C12Q

Abstract:
A diagnostic method for detecting mutation of a sample protein which disrupts binding to another target protein, comprising:i. isolating a gene encoding said sample protein from a sample of biopsied cells, wherein said sample of cells is suspected of containing mutational changes; ii. cloning said gene into a two hybrid assay to produce a host cell comprising (a) a reporter gene operably linked to a transcriptional regulatory sequence, (b) a first chimeric gene which encodes a first fusion protein, said first fusion protein including a target protein to which a wild-type form of said sample protein binds and to which binding of the cloned sample protein is to be assessed, (c) a second chimeric gene which encodes a second fusion protein including said cloned sample protein, wherein interaction of said first and second fusion proteins, if at all, is mediated by binding of said target protein and sample protein portions, and expression of said reporter gene is sensitive to interactions between said transcriptional regulatory sequence and a complex of said first and second fusion proteins;iii. measuring expression of said reporter gene; and iv. comparing the level of expression of said reporter gene to a level of expression in a two hybrid assay having a second fusion protein comprising a wild-type form of said sample protein instead of the cloned sample protein, wherein a decrease in the level of expression is indicative of a mutation to said gene which disrupts the ability of said sample protein to bind to another cellular protein, and which correlates to the presence of a disorder.