Comparison of two PCR-based methods and automated DNA sequencing for prop-1 genotyping in Ames dwarf mice

Arpad Gerstner, James H. DeFord, John Papaconstantinou

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

Ames dwarfism is caused by a homozygous single nucleotide mutation in the pituitary specific prop-1 gene, resulting in combined pituitary hormone deficiency, reduced growth and extended lifespan. Thus, these mice serve as an important model system for endocrinological, aging and longevity studies. Because the phenotype of wild type and heterozygous mice is undistinguishable, it is imperative for successful breeding to accurately genotype these animals. Here we report a novel, yet simple, approach for prop-1 genotyping using PCR-based allele-specific amplification (PCR-ASA). We also compare this method to other potential genotyping techniques, i.e. PCR-based restriction fragment length polymorphism analysis (PCR-RFLP) and fluorescence automated DNA sequencing. We demonstrate that the single-step PCR-ASA has several advantages over the classical PCR-RFLP because the procedure is simple, less expensive and rapid. To further increase the specificity and sensitivity of the PCR-ASA, we introduced a single-base mismatch at the 3′ penultimate position of the mutant primer. Our results also reveal that the fluorescence automated DNA sequencing has limitations for detecting a single nucleotide polymorphism in the prop-1 gene, particularly in heterozygotes.

Original languageEnglish (US)
Pages (from-to)37-44
Number of pages8
JournalMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume528
Issue number1-2
DOIs
StatePublished - Jul 25 2003

Keywords

  • Allele specific amplification
  • Ames dwarfism
  • Automated DNA sequencing
  • Restriction fragment length polymorphism
  • Single nucleotide polymorphism
  • prop-1 genotyping

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Health, Toxicology and Mutagenesis

Fingerprint Dive into the research topics of 'Comparison of two PCR-based methods and automated DNA sequencing for prop-1 genotyping in Ames dwarf mice'. Together they form a unique fingerprint.

  • Cite this