A cloning strategy for identification of genes containing trinucleotide repeat expansions.
Until today, nineteen trinucleotide repeat expansions larger than forty repeat copies have been found in the human genome. Of these, the CAG/CTG repeat is predominant motif with twelve loci identified, ten of which have been associated with the development of neurodegenerative diseases. We have developed a cloning approach which isolates disease genes containing trinucleotide repeat expansions. The method is based on size separation of genomic fragments, followed by subcloning and library hybridization with an oligonucleotide probe. Fractions and clones containing expanded repeats are identified by the repeat expansion detection (RED) method throughout the cloning procedure. Large family materials are not required and as little as 10 microg genomic DNA from a single individual is sufficient for this method. Using this strategy we have cloned two DNA fragments containing expanded repeats from two unrelated patients with a clinical diagnosis of cerebellar ataxia. Sequencing of the two fragments showed sequence identities with two disease genes, the Huntington gene and the ataxin 3 gene, respectively. The method should be adaptable to the cloning of any long repeat motif in any species. Furthermore the experimental steps can be performed in less than a month, making it very effective and time efficient to disease gene identification.
Yuan
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QP
Neurogenetics Unit, Department of Molecular Medicine, Karolinska Hospital, 17176 Stockholm, Sweden. qiu-ping.yuan@cmm.ki.se
Lindblad-Toh
K
K
Zander
C
C
Burgess
C
C
Durr
A
A
Schalling
M
M
D000067699:Ataxin-3; D002524:Cerebellar Ataxia / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics; D030561:Databases, Nucleic Acid; D005796:Genes / Q000235:genetics*; D006801:Humans; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D012097:Repressor Proteins; D017422:Sequence Analysis, DNA; D019680:Trinucleotide Repeat Expansion / Q000235:genetics*
C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D004247:DNA; C092341:ATXN3 protein, human; D000067699:Ataxin-3