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Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis
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Efficient method of genotyping ob/ob mice using high resolution melting analysis.

Alton G Sutter1, Arun P Palanisamy, Nichole Kurtz

  • 1Division of Transplant Surgery/Department of Surgery, Medical University of South Carolina, Charleston, South Carolina, United States of America.

Plos One
|November 16, 2013
PubMed
Summary

High Resolution Melting Analysis (HRM) offers a fast, cost-effective method for genotyping Lep(ob) mutations in obesity research. This technique accurately identifies mouse genotypes, crucial for maintaining breeding colonies and advancing obesity disease models.

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Area of Science:

  • Genetics and Genomics
  • Animal Models of Disease
  • Biotechnology

Background:

  • Obesity disease models are critical for research, with the ob/ob mouse being a well-established model.
  • Efficiently maintaining breeding colonies requires accurate identification of heterozygote Lep(ob) mice, as ob/ob mice are sterile.
  • Current genotyping methods for the Lep(ob) single nucleotide polymorphism (SNP) are often time-consuming, inconsistent, or expensive.

Purpose of the Study:

  • To develop and validate a fast, high-throughput, cost-effective, and consistent method for identifying the Lep(ob) mutation.
  • To establish a reliable genotyping technique for the Lep(ob) SNP in mouse models of obesity.

Main Methods:

  • Design and validation of primers for High Resolution Melting Analysis (HRM) of the Lep(ob) SNP.
  • Utilizing real-time PCR instrumentation for amplicon generation and subsequent HRM.

Main Results:

  • High Resolution Melting Analysis (HRM) successfully differentiated between ob/+, ob/ob, and wild-type (WT) genotypes.
  • Melting curve plots, normalized for fluorescence, clearly delineated the distinct genotypes.
  • Phenotypic confirmation supported the genotypic results obtained through HRM.

Conclusions:

  • HRM provides a sensitive, accurate, and closed-tube method for identifying the Lep(ob) mutation.
  • This technique leverages commonly available real-time PCR machines, making it accessible to most laboratories.
  • HRM offers advantages in terms of cost-effectiveness, reduced optimization, and assay consistency compared to traditional genotyping methods.