Identification of ATM mutations using extended RT-PCR and restriction endonuclease fingerprinting, and elucidation of the repertoire of A-T mutations in Israel

Shlomit Gilad, Kami Khosravi, Reli Harnik, Yael Ziv, Dganit Shkedy, Yaron Galanty, Moshe Frydman, Jacov Levi, Ozden Sanal, Luciana Chessa, Dominique Smcets, Yosef Shiloh, Anat Bar-Shira

Research output: Contribution to journalArticlepeer-review

Abstract

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 over-lapping 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.

Original languageEnglish
Pages (from-to)69-75
Number of pages7
JournalHuman Mutation
Volume11
Issue number1
DOIs
StatePublished - 1998

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