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


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.
Division of Transplant Surgery/Department of Surgery, Medical University of South Carolina, Charleston, South Carolina, United States of America.
Alton G
AG
Sutter
Arun P
AP
Palanisamy
Nichole
N
Kurtz
Demetri D
DD
Spyropoulos
Kenneth D
KD
Chavin
D000818:Animals; D006580:Genetic Carrier Screening; D060005:Genotyping Techniques; D006579:Heterozygote; D051379:Mice; D008810:Mice, Inbred C57BL; D008820:Mice, Obese; D020641:Polymorphism, Single Nucleotide; D044366:Transition Temperature; D015430:Weight Gain / Q000235:genetics