Repeat Analysis Pooled Isolation and Detection (RAPID) Cloning of Microsatellite Expansions

Microsatellite repeat expansions have been shown to cause a number of neurodegenerative diseases (1 ). Most of the disease genes identified to date involve the expansion of a trinucleotide repeat motif, but recently tetra- and pentanucleotide repeat expansions have been shown to cause myotonic dystrophy type 2 (DM2) and spinocerebellar ataxiatype 10 (SCA10), respectively (2 ,3 ). Most microsatellite diseases are characterized by the presence of anticipation, or a decrease in the age of onset in consecutive generations due to the tendency of the unstable repeat tract to lengthen when passed from one generation to the next (1 ,4 ,5 ). In addition, the involvement of trinucleotide repeat expansions in a number of other diseases including schizophrenia (6 ) and bipolar affective disorder (7 ,8 ) has been suggested both by the presence of anticipation and by Repeat Expansion Detection (RED) analysis (9 ,10 ). The involvement of trinucleotide expansions in these diseases, however, can only be conclusively confirmed by the isolation of the expansions present in these populations and detailed analysis to assess each expansion as a possible pathogenic mutation. We previously described a novel procedure to quickly isolate expanded trinucleotide repeats and the corresponding flanking nucleotide sequence directly from small amounts of genomic DNA using a process of Repeat Analysis, Pooled Isolation, and Detection of individual clones containing expanded trinucleotide repeats (RAPID cloning) (11 ). We used this technology to clone the pathogenic SCA7 and SCA8 CAG/CTG repeat expansions from banked DNA samples from single individuals affected with ataxia (11 –13 ).