Identification of mutations from phenotype-driven ENU mutagenesis in mouse chromosome 7.
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.
Culiat
Cymbeline T
CT
Life Sciences Division, Oak Ridge National Laboratory, Bethel Valley Road, P.O. Box 2008, Oak Ridge, Tennessee, 37831-6445, USA. culiatct@ornl.gov
Klebig
Mitchell L
ML
Liu
Zhaowei
Z
Monroe
Heidi
H
Stanford
Beverly
B
Desai
Jayashree
J
Tandan
Samvit
S
Hughes
Lori
L
Kerley
Marilyn K
MK
Carpenter
Donald A
DA
Johnson
Dabney K
DK
Rinchik
Eugene M
EM
Li
Qingbo
Q
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
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