PUBLICATION

A genotyping method combining primer competition PCR with HRM analysis to identify point mutations in Duchenne animal models

Authors
Lasa-Fernandez, H., Mosqueira-Martín, L., Alzualde, A., Lasa-Elgarresta, J., Vallejo-Illarramendi, A.
ID
ZDB-PUB-201020-15
Date
2020
Source
Scientific Reports   10: 17224 (Journal)
Registered Authors
Alzualde, Ainhoa
Keywords
none
MeSH Terms
  • Genotyping Techniques/methods*
  • Drug Evaluation, Preclinical
  • Birefringence
  • Polymerase Chain Reaction/methods*
  • Mice, Inbred mdx
  • Muscular Dystrophy, Duchenne/genetics*
  • Mice, Inbred C57BL
  • Molecular Diagnostic Techniques/methods*
  • Animals
  • Disease Models, Animal*
  • Point Mutation*
  • Sensitivity and Specificity
  • Zebrafish
(all 13)
PubMed
33057138 Full text @ Sci. Rep.
Abstract
Dystrophin-null sapje zebrafish is an excellent model for better understanding the pathological mechanisms underlying Duchenne muscular dystrophy, and it has recently arisen as a powerful tool for high-throughput screening of therapeutic candidates for this disease. While dystrophic phenotype in sapje larvae can be easily detected by birefringence, zebrafish genotyping is necessary for drug screening experiments, where the potential rescue of larvae phenotype is the primary outcome. Genotyping is also desirable during colony husbandry since heterozygous progenitors need to be selected. Currently, sapje zebrafish are genotyped through techniques involving sequencing or multi-step PCR, which are often costly, tedious, or require special equipment. Here we report a simple, precise, cost-effective, and versatile PCR genotyping method based on primer competition. Genotypes can be resolved by standard agarose gel electrophoresis and high-resolution melt assay, the latter being especially useful for genotyping a large number of samples. Our approach has shown high sensitivity, specificity, and reproducibility in detecting the A/T point mutation in sapje zebrafish and the C/T mutation in the mdx mouse model of Duchenne. Hence, this method can be applied to other single nucleotide substitutions and may be further optimized to detect small insertions and deletions. Given its robust performance with crude DNA extracts, our strategy may be particularly well-suited for detecting single nucleotide variants in poor-quality samples such as ancient DNA or DNA from formalin-fixed, paraffin-embedded material.
Genes / Markers
Figures
No images available
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Expression
No data available
Phenotype
Fish Conditions Stage Phenotype Figure
dmdta222a/ta222astandard conditionsDay 6
1 - 1 of 1
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Mutations / Transgenics
Allele Construct Type Affected Genomic Region
ta222a
    		Point Mutation
    1 - 1 of 1
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    Human Disease / Model
    Human Disease Fish Conditions Evidence
    Duchenne muscular dystrophydmdta222a/ta222astandard conditionsTAS
    1 - 1 of 1
    Show
    Sequence Targeting Reagents
    No data available
    Fish
    Fish
    dmdta222a/ta222a
    1 - 1 of 1
    Show
    Antibodies
    Orthology
    Engineered Foreign Genes
    No data available
    Mapping
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    Citation
    Lasa-Fernandez, H., Mosqueira-Martín, L., Alzualde, A., Lasa-Elgarresta, J., Vallejo-Illarramendi, A. (2020) A genotyping method combining primer competition PCR with HRM analysis to identify point mutations in Duchenne animal models. Scientific Reports. 10:17224.