Multiplex DNA amplification and solid-phase direct sequencing for mutation analysis at the hprt locus in Chinese hamster cells.

TitleMultiplex DNA amplification and solid-phase direct sequencing for mutation analysis at the hprt locus in Chinese hamster cells.
Publication TypeJournal Article
Year of Publication1993
AuthorsZu, Z, Yu, Y, Gibbs, RA, Caskey, CT, Hsie, AW
JournalMutat Res
Volume288
Issue2
Pagination237-48
Date Published1993 Aug
ISSN0027-5107
KeywordsAnimals, Base Sequence, CHO Cells, Chromosome Mapping, Cricetinae, DNA Mutational Analysis, Hypoxanthine Phosphoribosyltransferase, Molecular Sequence Data, Mutation, Polymerase Chain Reaction, Sequence Analysis, DNA
Abstract

We report here the development of multiplex in vitro DNA amplification and solid-phase direct exon sequencing for the analysis of mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster cells. 18 representative HPRT-deficient mutants, derived either spontaneously, or after exposure to UV light or ionizing radiation, were analyzed. All 9 hprt exons were simultaneously amplified via the polymerase chain reaction (PCR) for rapid deletion detection. 5 mutants involve single- or multiple-exon deletions. Altered multiplex PCR patterns were detected in mutants Bsp-040, Bsp-065 and BGR-606. Subsequent direct sequence analysis reveals that Bsp-040 and Bsp-065 carry a 52-bp and a 13-bp intragenic DNA deletion in exon 3, respectively. BGR-606 contains a 223-bp insertion accompanied by a 10-bp deletion of intron sequence within exon 4 fragment. Other subtle DNA alterations identified by direct exon sequence analysis include single-base substitutions, small deletions and insertions, and RNA splicing mutations.

Alternate JournalMutat. Res.
PubMed ID7688083