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Identification of ATM mutations using extended RT‐PCR and restriction endonuclease fingerprinting, and elucidation of the repertoire of A‐T mutations in Israel
Author(s) -
Gilad Shlomit,
Khosravi Rami,
Harnik Reli,
Ziv Yael,
Shkedy Dganit,
Galanty Yaron,
Frydman Moshe,
Levi Jacov,
Sanal Ozden,
Chessa Luciana,
Smeets Dominique,
Shiloh Yosef,
BarShira Anat
Publication year - 1998
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/(sici)1098-1004(1998)11:1<69::aid-humu11>3.0.co;2-x
Subject(s) - biology , genetics , open reading frame , restriction enzyme , rna splicing , microbiology and biotechnology , gene , population , mutation , rna , peptide sequence , demography , sociology
Ataxia‐telangiectasia (A‐T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, and radiation sensitivity. The responsible gene, ATM, has an extensive genomic structure and encodes a large transcript with a 9.2 kb open reading frame (ORF). A‐T mutations are extremely variable and most of them are private. We streamlined a high throughput protocol for the search for ATM mutations. The entire ATM ORF is amplified in a single RT‐PCR step requiring a minimal amount of RNA. The product can serve for numerous nested PCRs in which overlapping portions of the ORF are further amplified and subjected to restriction endonuclease fingerprinting (REF) analysis. Splicing errors are readily detectable during the initial amplification of each portion. Using this protocol, we identified 5 novel A‐T mutations and completed the elucidation of the molecular basis of A‐T in the Israeli population. Hum Mutat 11:69–75, 1998. © 1998 Wiley‐Liss, Inc.