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増大CAGリピートの高感度検出法 : 2次元電気泳動を用いたdirect identification of repeat expansion and cloning technique(DIRECT)の開発

フォーマット:
論文(リポジトリ)
責任表示:
池内, 健
出版情報:
新潟医学会, 2000-05
掲載情報:
新潟医学会雑誌 = 新潟医学会雑誌
ISSN:
00290440  CiNii Articles  Webcat Plus  JAIRO
著者名:
池内, 健  
巻:
114
通号:
5
開始ページ:
186
終了ページ:
199
バージョン:
publisher
概要:
Recent discoveries of genes containing CAG/CTG repeats as the causative genes in hereditary neurodegenerative diseases suggest that trinucleotide repeat expansions could also be associated with a number of other neurological disorders. The CAG repeats in the genes of normal individuals are generally less than 35 repeat units in size, whereas the pathologically expanded alleles contain repeats approximately in excess of 40 units. We initially developed a novel method, direct identification of repeat expansion and cloning technique(DIRECT), which allows selective detection of CAG repeats containing expanded alleles exceeding 40 CAG/CTG repeat units, by high-stringent hybridization condition. By applying DIRECT, we cloned the SCA2 and Dirl genes, both of which contain CAG/CTG repeats larger than 40 repeat units. Discovery of the mutation in spinocerebellar ataxia type 6(SCA 6), in which the expanded CAG repeats contain in the range of 21-26 units suggests that the CAG-repeat diseases are not always associated with genes that carry expanded alleles larger than 40 repeat units. In the high-stringent condition of hybridization in the original DIRECT, however, mildly expanded alleles containing smaller than 40 repeat units, as in the SCA 6 mutation, could not be detected. Thus, I tried to establish hybridization conditions which allow detection of mildly expanded alleles with the cutoff point at around 20 repeat units by increasing the ionic strength of SSPE. Although the expanded allele in SCA 6 patients was specifically detected in the low-stringent hybridization condition(3.25×SSPE), multiple bands, which may hamper detection of pathologically expanded allele, also appeared. To achieve high resolution of genomic segments detected by the probe, I applied in gel digestion and two-dimensional electrophoresis to improve the power of detection of expanded CAG repeats by DIRECT with low-stringent hybridization. Sixteen to 38 discrete spots, supposed as representing CAG/CTG repeats larger than 20 repeat units, are observed, some of which are polymorphic among normal individuals. In the model system using genomic DNA from a patient with dentatorubral-pallidoluysian atrophy(DRPLA), an expanded allele containing 68 repeat units was discretely detected as an extra spot. I here describe the development of a new technique, two-dimensional DIRECT(2D-DIRECT), which could become an efficient and powerful method for identifying novel genes of diseases associated with wide range of expanded CAG/CTG repeats. 続きを見る
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