Comparison of DNA extraction methods for genotyping equine histidine-rich glycoprotein insertion/deletion polymorphisms using oral mucosa swabs and feces.
Abstract: Previously, we demonstrated unique insertion/deletion polymorphisms of equine histidine-rich glycoprotein with five genotypes composed of 45-bp or 90-bp deletions in the histidine-rich region of in Thoroughbred horses. Although leukocytes are typically used to collect DNA for genotyping, blood sampling from animals is sometimes difficult and invasive. Moreover, the method for extracting DNA from blood leukocytes involves complicated steps and must be performed soon after blood sampling for sensitive gene analysis. In the present study, we performed genotyping using DNA, isolated from oral mucosa swabs collected by rubbing the mucosa on the underside of the upper lip of horses and 100 mg of freshly excreted feces obtained by scraping their surface. In the present study, we performed genotyping using DNA isolated from oral mucosa swabs and feces of horses (18 Thoroughbreds, 17 mixed breeds, 2 warm bloods), and compared the accuracy of this method with that of the method using DNA from leukocytes. The DNA derived from oral mucosa swabs was sufficient in quantity and quality for genotyping. However, DNA derived from fecal samples requires a more sensitive detection system because of contamination with non-horse DNA, and the test quality is low. Collection of oral mucosa swabs is less invasive than blood sampling; further, oral swabs can be stored for a longer period in a specified high-quality solution. Therefore, collecting DNA samples from oral mucosa swabs is recommended for the genetic analysis of not only horses but also other animals that are not accustomed to humans.
© 2024 The Author(s).
Publication Date: 2024-05-23 PubMed ID: 38947185PubMed Central: PMC11214520DOI: 10.1016/j.vas.2024.100361Google Scholar: Lookup
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Summary
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The research explores different methods for extracting DNA and genotyping a specific equine gene using non-invasive samples such as oral swabs and feces. The study found that collecting DNA samples from oral mucosa swabs was the most ideal method due to its less invasive nature, longer storage period and quality of genetic analysis results.
Objective and Background:
- The objective of the research paper was to explore alternative and less invasive methods for genotyping equine histidine-rich glycoprotein insertion/deletion polymorphisms aside from blood sampling which is typically used but is often invasive and complicated.
- This study comes after the researchers were able to demonstrate distinct polymorphisms of the equine histidine-rich glycoprotein within five genotypes of Thoroughbred horses, hence their interest in novel genotyping methods.
Methods and Procedures:
- The researchers performed genotyping using DNA samples that were extracted from oral mucosa swabs from the upper lip mucosa and 100 mg of freshly excreted feces obtained by scraping the surface.
- The genotyping was performed on DNA samples from a diverse set of horses including 18 Thoroughbreds, 17 mixed breeds, and 2 warm bloods. The results were then compared with the traditional method that employs DNA from leukocytes.
Findings:
- The results showed that DNA extraction from oral mucosa swabs yielded both sufficient quantity and quality for effective genotyping.
- On the other hand, DNA extracted from fecal samples was found to require a more sensitive detection system due to the contamination from non-horse DNA. This resulted in lower test quality from this method.
Conclusion:
- The researchers concluded that DNA sample collection from oral mucosa swabs is the preferred method for genotyping equine histidine-rich glycoprotein insertion/deletion polymorphisms due to its less invasive nature and the higher quality of results that can be obtained.
- They also pointed out that oral swabs can be stored longer in a specified high-quality solution, making them a practical consideration for the genetic analysis of not only horses but also other animals unaccustomed to human interaction.
Cite This Article
APA
Muko R, Ojima Y, Matsuda H, Toishi Y, Oikawa MA, Shin T, Sato H, Tanaka A.
(2024).
Comparison of DNA extraction methods for genotyping equine histidine-rich glycoprotein insertion/deletion polymorphisms using oral mucosa swabs and feces.
Vet Anim Sci, 25, 100361.
https://doi.org/10.1016/j.vas.2024.100361 Publication
Researcher Affiliations
- Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, Tokyo, Japan.
- Laboratory of Comparative Animal Medicine, Division of Animal Life Science, Institute of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan.
- Laboratory of Comparative Animal Medicine, Division of Animal Life Science, Institute of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan.
- Shadai Stallion Station, Shadai Corporation, Hokkaido, Japan.
- Diagnostic and Research Laboratory, Equine Veterinary Medical Center, Education City, Doha, Qatar.
- Department of Veterinary Anatomy, College of Veterinary Medicine and Veterinary Medical Research Institute, Jeju National University, Jeju, South Korea.
- Stewards Department, Race Integrity Section, Japan Racing Association, Tokyo, Japan.
- Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, Tokyo, Japan.
- Laboratory of Comparative Animal Medicine, Division of Animal Life Science, Institute of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan.
Conflict of Interest Statement
The authors declare no potential conflicts of interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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