Identification of mutations from phenotype-driven ENU mutagenesis in mouse chromosome 7.
Mamm Genome
; 16(8): 555-66, 2005 Aug.
Article
em En
| MEDLINE
| ID: mdl-16180137
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Análise Mutacional de DNA
/
Mutagênese
/
Mutação Puntual
/
Cromossomos de Mamíferos
/
Etilnitrosoureia
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Revista:
Mamm Genome
Ano de publicação:
2005
Tipo de documento:
Article