Detection and isolation of trinucleotide repeat expansions using the RED method.
To facilitate identification of disease genes containing an expanded trinucleotide repeat, a repeat expansion detection (RED) and gene cloning system was established. The RED method was developed to enable detection of expanded trinucleotide repeat sequences in any DNA sample from any species without prior knowledge of the DNA sequences flanking the repeat. The DNA to be tested is used as a template for a repeat oligonucleotide to anneal and ligate in a two-step cycling procedure. After hundreds of annealing/ligation cycles, a large amount of oligonucleotide multimers is accumulated. The longest multimer represents the largest repeat expansion in the genome tested. The gene isolation strategy is based on size separation of genomic fragments, followed by subcloning and library hybridization with an oligonucleotide probe. The expanded trinucleotide repeat is identified throughout the procedure using the RED method. Using this approach, two disease genes, the Huntington's disease gene and the MJD/SCA3 gene, were cloned. This cloning strategy should be applicable to isolation of any DNA fragment containing large trinucleotide repeat expansions in any species.
Yuan
Qiu-Ping
QP
Division of Hematology, University of Wisconsin, Medical Science Center, Madison, USA.
Schalling
Martin
M
D001345:Autoradiography; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005821:Genetic Techniques; D018911:Trinucleotide Repeats / Q000235:genetics*