Date of Award:

8-2023

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Animal, Dairy, and Veterinary Sciences

Committee Chair(s)

Lee F. Rickords

Committee

Lee F. Rickords

Committee

Kerry A. Rood

Committee

Matthew Garcia

Abstract

PCR sex determination assays must be reliable and cost effective due to the frequent and integral use of these assays in biological research and the animal production industry. Thus, the design of proof of a primer pair with a built-in control is warranted to not only bypass the extra cost of a multiplex reaction, but also to prevent anomalous results that have been documented with other primer pairs.

The objective of this study was to design primer pairs with built in PCR amplification control to identify sex in Equus caballus (domestic horse), Homo sapiens (humans), Macaca mulatta (rhesus macaque), and Sus scrofa (domestic pig) DNA samples. The procedures utilized in this study were to first align the DDX3X gene with the Y chromosome homolog and create primer pairs to flank Y chromosome specific indels with each species. PCR with gel electrophoresis results showed confirmation of the hypothesis that the primer pairs can be used to accurately identify separate X and Y chromosome specific sequences via species-specific single primer pairs in Equus caballus, Homo sapiens, Macaca mulatta, and Sus scrofa genomic samples. Additionally, in silico and qualitative gel analyses were completed to assess the efficacy of the Equus caballus specific primers in alternative species, which yielded no results.

Summarily, results concluded that there are suitable indel regions for PCR amplification in Equus caballus, Homo sapiens, Macaca mulatta, and Sus scrofa. The results presented herein represent a meaningful contribution to the field from this methodology, these indel regions can be identified via the designed primers and utilized in an efficient, quick, and cost-effective way to identify sex without the need for multiplex or the risk of false identification.

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