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PLoS One. 2013 Nov 13;8(11):e78840. doi: 10.1371/journal.pone.0078840. eCollection 2013.

Efficient method of genotyping ob/ob mice using high resolution melting analysis.

Author information

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

Abstract

OBJECTIVE:

Direct health care costs of obesity continue to grow throughout the world and research on obesity disease models are on the rise. The ob/ob mouse is a well-characterized model of obesity and associated risk factors. Successful breeding and backcrossing onto different backgrounds are essential to create knockout models. Ob/ob mice are sterile and heterozygotes must be identified by genotyping to maintain breeding colonies. Several methods are employed to detect the ob mutant allele, a single nucleotide polymorphism (SNP). Gel based methods are time consuming and inconsistent, and non-gel based assays rely upon expensive and complex reagents or instruments. A fast, high-throughput, cost effective, and consistent method to identify Lep(ob) mutation is much needed.

DESIGN AND METHODS:

Primers to produce an amplicon for High Resolution Melting Analysis (HRM) of the Lep(ob) SNP were designed and validated.

RESULTS:

Fluorescence normalized high resolution melting curve plots delineated ob/+, ob/ob, and WT genotypes. Genotypes were also confirmed phenotypically.

CONCLUSIONS:

HRM of the Lep(ob) SNP allows closed-tube identification of the Lep(ob) mutation using a real-time PCR machine now common to most labs/departments. Advantages of this method include assay sensitivity/accuracy, low cost dyes, less optimization, and cost effectiveness as compared to other genotyping techniques.

PMID:
24236058
PMCID:
PMC3827293
DOI:
10.1371/journal.pone.0078840
[Indexed for MEDLINE]
Free PMC Article

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