title: Identification of mutations from phenotype-driven ENU mutagenesis in mouse chromosome 7.
abstract: We have used the new high-throughput mutation-scanning technique temperature-gradient capillary electrophoresis (TGCE) for the identification of point mutations induced by N-ethyl-N-nitrosourea (ENU) in the mouse genome. TGCE detects the presence of heteroduplex molecules formed between a wild-type gene segment and the corresponding homologous segment containing an induced mutation or a naturally occurring single nucleotide polymorphism (SNP). Partially denatured heteroduplex molecules are resolved from homoduplexes by virtue of their differential mobilities during capillary electrophoresis conducted in a finely controlled temperature gradient. Simultaneous heteroduplex analysis of 96 amplicons ranging from 150 to 600 bp in size is achieved in approximately 45 min without the need for predetermining the melting profile of each fragment. Initially, we exploited known mouse mutations to develop TGCE protocols for analyzing unpurified PCR samples amplified from crude tail-DNA preparations. TGCE was then applied to the rapid identification of three new ENU-induced mutations recovered from regional mutagenesis screens of a segment of mouse Chromosome 7. Enzyme assays and quantitative reverse transcription-PCR (qRT-PCR) methods validated these new mutations. Our data demonstrate that rapid mutation scanning with TGCE, followed by sequence verification only of detected positives, is an efficient approach to the identification of point mutations in the mouse genome.
authors.affiliation: Life Sciences Division, Oak Ridge National Laboratory, Bethel Valley Road, P.O. Box 2008, Oak Ridge, Tennessee, 37831-6445, USA. culiatct@ornl.gov
authors.forename: Cymbeline T
authors.initials: CT
authors.lastname: Culiat
authors.forename: Mitchell L
authors.initials: ML
authors.lastname: Klebig
authors.forename: Zhaowei
authors.initials: Z
authors.lastname: Liu
authors.forename: Heidi
authors.initials: H
authors.lastname: Monroe
authors.forename: Beverly
authors.initials: B
authors.lastname: Stanford
authors.forename: Jayashree
authors.initials: J
authors.lastname: Desai
authors.forename: Samvit
authors.initials: S
authors.lastname: Tandan
authors.forename: Lori
authors.initials: L
authors.lastname: Hughes
authors.forename: Marilyn K
authors.initials: MK
authors.lastname: Kerley
authors.forename: Donald A
authors.initials: DA
authors.lastname: Carpenter
authors.forename: Dabney K
authors.initials: DK
authors.lastname: Johnson
authors.forename: Eugene M
authors.initials: EM
authors.lastname: Rinchik
authors.forename: Qingbo
authors.initials: Q
authors.lastname: Li
mesh_terms: D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D033481:Chromosomes, Mammalian / Q000235:genetics*; D004252:DNA Mutational Analysis; D019075:Electrophoresis, Capillary; D005038:Ethylnitrosourea / Q000494:pharmacology*; D020180:Heteroduplex Analysis; D007527:Isoenzymes / Q000235:genetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D016296:Mutagenesis / Q000187:drug effects*; D010105:Oxygenases / Q000235:genetics; D010641:Phenotype; D017354:Point Mutation / Q000235:genetics*; D000685:Serum Amyloid A Protein / Q000235:genetics
chemical_list: D007527:Isoenzymes; C076498:Saa3 protein, mouse; D000685:Serum Amyloid A Protein; D007770:L-Lactate Dehydrogenase; C409667:lactate dehydrogenase 1; D010105:Oxygenases; C426838:fatty acid alpha-hydroperoxylase; D005038:Ethylnitrosourea