Comparison of two PCR-based methods and automated DNA sequencing for prop-1 genotyping in Ames dwarf mice.
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.
Gerstner
Arpad
A
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77550, USA.
DeFord
James H
JH
Papaconstantinou
John
J
D000818:Animals; D001331:Automation; D005838:Genotype; D018398:Homeodomain Proteins / Q000235:genetics*; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA
D018398:Homeodomain Proteins; C102134:Prophet of Pit-1 protein