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

Zhidong Xu, Yongjia Yu, Richard A. Gibbs, C. Thomas Caskey, Abraham W. Hsie

Research output: Contribution to journalArticle

26 Citations (Scopus)

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, adn RNA splicing mutations.

Original languageEnglish (US)
Pages (from-to)237-248
Number of pages12
JournalMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume288
Issue number2
DOIs
StatePublished - 1993

Fingerprint

Hypoxanthine Phosphoribosyltransferase
Cricetulus
Exons
Mutation
DNA
Sequence Analysis
RNA Splicing
Sequence Deletion
Multiplex Polymerase Chain Reaction
Ultraviolet Rays
Ionizing Radiation
Introns
Polymerase Chain Reaction

Keywords

  • Deletion screening
  • Direct DNA sequencing
  • Molecular mutagenesis
  • Multiplex PCR
  • RNA splicing errors

ASJC Scopus subject areas

  • Molecular Biology
  • Health, Toxicology and Mutagenesis

Cite this

Multiplex DNA amplification and solid-phase direct sequencing for mutation analysis at the hprt locus in Chinese hamster cells. / Xu, Zhidong; Yu, Yongjia; Gibbs, Richard A.; Thomas Caskey, C.; Hsie, Abraham W.

In: Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 288, No. 2, 1993, p. 237-248.

Research output: Contribution to journalArticle

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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, adn RNA splicing mutations.",
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AU - Hsie, Abraham W.

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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.

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