Development of a Single Reaction PCR Genotyping Assay for Two Separate Gene Loci in Mice

Abstract

There are many variables to consider when optimizing a polymerase chainreaction (PCR) assay for a specific gene—from methods of DNA extraction to choosing an effective combination of primers. A multiplex PCR assay analyzing two genes in the same reaction may require additional optimization. The objective of this experiment was to optimize a single PCR assay to effectively genotype C57BL/6 transgenic mouse strains in which two genes have been modified. By removing a portion of the Es1 gene, the expression of serum carboxylesterase was interrupted, resulting in serum carboxylesterase knock out (Es1 KO) mice. In another location, the gene expressing mouse acetylcholinesterase was replaced by a gene expressing human acetylcholinesterase, resulting in a human acetylcholinesterase knock in (AChEKI) mouse strain. There are several possible genotypes for each mouse strain: for Es1 KO mice the possibilities are wild type (WT +/+), knock out (KO −/−), or heterozygous (Het +/−), and for AChEKI mice the possibilities are human (H/H), mouse (M/M), or heterozygous (M/H). Optimization of the PCR genotyping assays for these strains was achieved by altering the method of DNA extraction, annealing temperatures, and testing multiple primer sets. Initially, DNA was extracted from mouse fecal matter, but this method was later determined to produce unreliable results in the subsequent PCR assays. DNA extracted from tail tissue was found to be much more reliable. Utilizing tail tissue DNA, a single pair of primers was found that could differentiate WT, KO, and Het alleles in the Es1 KO strain. The optimal annealing temperature for these primers was determined using a temperature gradient experiment. Primers for genotyping the AChE KI strain were selected and optimized in a similar manner. Finally, a multiplex PCR assay was developed allowing for the simultaneous determination of the genotype of both genes in mice.Support or Funding Information*This research was supported in part by an appointment to the Postgraduate Research Participation Program at the U.S. Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U. S. Army Medical Research and Materiel Command.The experimental protocol was approved by the Animal Care and Use Committee at the United States Army Medical Research Institute of Chemical Defense and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011), and the Animal Welfare Act of 1966 (P.L. 89‐544), as amended.Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division.The views expressed in this abstract are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.