|Title||Multiplex DNA amplification and solid-phase direct sequencing for mutation analysis at the hprt locus in Chinese hamster cells.|
|Publication Type||Journal Article|
|Year of Publication||1993|
|Authors||Zu, Z, Yu, Y, Gibbs, RA, Caskey, CT, Hsie, AW|
|Date Published||1993 Aug|
|Keywords||Animals, Base Sequence, CHO Cells, Chromosome Mapping, Cricetinae, DNA Mutational Analysis, Hypoxanthine Phosphoribosyltransferase, Molecular Sequence Data, Mutation, Polymerase Chain Reaction, Sequence Analysis, DNA|
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 Journal||Mutat. Res.|