Modified rapid expansion detection method to analyze CAG/CTG repeat expansions

摘要

Trinucleotide repeat expansions (TREs) have been associated with several genetic neurological and neuromuscular disorders including Huntington disease, Fragile X syndrome, myotonic dystrophy, and Friedreich ataxia (1, 11,12). Among trinucleotide repeats, the expansion of CAG/CTG has been studied most extensively because the expansion of this repeat is found to be associated very frequently in neurological and neuromuscular disorders. Repeat expansion detection (RED) has been used widely to identify and locate TREs in the human genome. The RED technique was introduced by Schalling et al. (8) and modified by other investigators (5,13,14). This method allows the detection of expanded repeats without prior knowledge of the location of the repeats or the flanking sequences. In a RED reaction, adjacent phosphorylated short oligonucleotides that anneal to TREs containing genomic DNA template are ligated with a thermostable DNA ligase (Ampligase~(R); Epicentre, Madison, WI, USA). These ligated oligonucleotides are then electrophoresed on a gel, transferred to nylon membrane, and visualized by hybridization with a radiolabeled probe. The details of the published protocol are as follows. Phosphorylation of oligonucleotides is carried out using ATP in the presence of T4 polynucleotide kinase. The ligation reactions are performed in 400-500 cycles of 20 s ligation at 65 deg C-75 deg C, according to the length of oligonucleotides used, and 10 s denaturing at 95 deg C. This ligation reaction is linear compared to an exponential PCR. In each cycle of the ligation reaction, only one copy of the ligated oligonucleotides is produced; thus, the RED product yield is very low. Therefore, compared to PCR-based methods, a large amount of starting genomic DNA template is required in a RED analysis.