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 Citations (Scopus)

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

Fingerprint

DNA Sequence Analysis
Polymerase Chain Reaction
Alleles
Restriction Fragment Length Polymorphisms
Genotyping Techniques
Fluorescence
Dwarfism
Heterozygote
Genes
Breeding
Single Nucleotide Polymorphism
Nucleotides
Genotype
Phenotype
Sensitivity and Specificity
Mutation
Growth

Keywords

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

ASJC Scopus subject areas

  • Health, Toxicology and Mutagenesis
  • Molecular Biology

Cite this

Comparison of two PCR-based methods and automated DNA sequencing for prop-1 genotyping in Ames dwarf mice. / Gerstner, Arpad; DeFord, James H.; Papaconstantinou, John.

In: Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 528, No. 1-2, 25.07.2003, p. 37-44.

Research output: Contribution to journalArticle

@article{1ccb815e588e4cc4a677d35b6ffb3c84,
title = "Comparison of two PCR-based methods and automated DNA sequencing for prop-1 genotyping in Ames dwarf mice",
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.",
keywords = "Allele specific amplification, Ames dwarfism, Automated DNA sequencing, prop-1 genotyping, Restriction fragment length polymorphism, Single nucleotide polymorphism",
author = "Arpad Gerstner and DeFord, {James H.} and John Papaconstantinou",
year = "2003",
month = "7",
day = "25",
doi = "10.1016/S0027-5107(03)00080-0",
language = "English (US)",
volume = "528",
pages = "37--44",
journal = "Mutation Research",
issn = "0027-5107",
publisher = "Elsevier",
number = "1-2",

}

TY - JOUR

T1 - Comparison of two PCR-based methods and automated DNA sequencing for prop-1 genotyping in Ames dwarf mice

AU - Gerstner, Arpad

AU - DeFord, James H.

AU - Papaconstantinou, John

PY - 2003/7/25

Y1 - 2003/7/25

N2 - 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.

AB - 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.

KW - Allele specific amplification

KW - Ames dwarfism

KW - Automated DNA sequencing

KW - prop-1 genotyping

KW - Restriction fragment length polymorphism

KW - Single nucleotide polymorphism

UR - http://www.scopus.com/inward/record.url?scp=0038107799&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0038107799&partnerID=8YFLogxK

U2 - 10.1016/S0027-5107(03)00080-0

DO - 10.1016/S0027-5107(03)00080-0

M3 - Article

C2 - 12873721

AN - SCOPUS:0038107799

VL - 528

SP - 37

EP - 44

JO - Mutation Research

JF - Mutation Research

SN - 0027-5107

IS - 1-2

ER -