alexa Single cell analysis of mutations in the APC gene.
Engineering

Engineering

Journal of Biosensors & Bioelectronics

Author(s): Mayer V, Schoen U, HolinskiFeder E, Koehler U, Thalhammer S

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Abstract INTRODUCTION: Mutation analysis of inherited monogenic diseases is an important aspect of preimplantation genetic diagnosis. Familial adenomatous polyposis of the colon is an autosomal dominant inherited disorder caused by mutations in the tumor suppressor gene adenomatous polyposis coli (APC). A characteristic of this severe disease is the development of thousands of polyps in the colon which untreated evolve into malignant colon carcinomas. Here we present a novel approach for the establishment of mutation detection in the APC gene in single cells applicable for preimplantation genetic diagnosis. METHODS: Single fixed lymphocytes were isolated via laser microdissection and transferred to reaction centers of planar chemically structured glass slides via a recently developed horizontal low-pressure single particle adsorbing transfer system (SPATS). A multiplex nested polymerase chain reaction protocol in a 1-microl reaction volume, followed by sequencing and fragment length analysis was applied in order to detect mutations in the APC gene. RESULTS: Reliable isolation and transfer of single lymphocytes was demonstrated. High amplification efficiency and low allelic drop out (ADO) rates for polymorphic microsatellite markers and mutation specific amplification products of various mutations in the APC gene were obtained from fixed single cells. CONCLUSIONS: This novel nanotechnological downscaling approach enables a reliable validation of genetic testing using diploid single lymphocytes, and will open a wide range of single cell diagnostics. This article was published in Fetal Diagn Ther and referenced in Journal of Biosensors & Bioelectronics

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