TY - JOUR
T1 - Multiplex DNA amplification and solid-phase direct sequencing for mutation analysis at the hprt locus in Chinese hamster cells
AU - Xu, Zhidong
AU - Yu, Yongjia
AU - Gibbs, Richard A.
AU - Thomas Caskey, C.
AU - Hsie, Abraham W.
N1 - Funding Information:
We thank Dr. Belinda J.F. Rossiter for kindly providing the hprt exon flanking sequences before publication and Drs. Joy A. Ahlgren-Beckendorf and Ronald C. Porter for review of the manuscript. This work was supported in part by NIEHS Small Business Innovative grant to LGS/Laboratories for Genetic Services, Inc., Houston, Texas, and The John Sealy Memorial Endowment fund to AWH. ZX and YY were recipients of fellowships from American Lung Association/San Jacinto Area. RAG is a recipient of the Muscular Dystrophy Association' Robert G. Sampson Distinguished Research Fol-lowship and CTC is an Investigator of the Howard Hughes Medical Institute.
PY - 1993/8
Y1 - 1993/8
N2 - 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, adn RNA splicing mutations.
AB - 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, adn RNA splicing mutations.
KW - Deletion screening
KW - Direct DNA sequencing
KW - Molecular mutagenesis
KW - Multiplex PCR
KW - RNA splicing errors
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U2 - 10.1016/0027-5107(93)90090-3
DO - 10.1016/0027-5107(93)90090-3
M3 - Article
C2 - 7688083
AN - SCOPUS:0027292126
SN - 0027-5107
VL - 288
SP - 237
EP - 248
JO - Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
JF - Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
IS - 2
ER -