Optimizing PCR for Mouse Genotyping: Recommendations for Reliable, Rapid, Cost Effective, Robust and Adaptable to High-Throughput Genotyping Protocol for Any Type of Mutation

Curr Protoc Mouse Biol. 2019 Dec;9(4):e65. doi: 10.1002/cpmo.65.


Genotyping consists of searching for a DNA sequence variation localized at a well-defined locus in the genome. It is an essential step in animal research because it allows the identification of animals that will be bred to generate and maintain a colony, euthanized to control the available space in the animal facility, or used in experiment protocols. Here we describe polymerase chain reaction (PCR) genotyping protocols for fast, sensitive, easy, and cost-effective characterization of mouse genotype. We discuss optimization of parameters to improve the reliability of each assay and propose recommendations for enhancing reproducibility and reducing the occurrence of inconclusive genotyping. All steps required for efficient genotyping are presented: tissue collection; sample verification and direct DNA lysis; establishment of a robust genotyping strategy with reliable, rapid, and cost-effective assays; and finally, transition to high-throughput automatized PCR, including mix miniaturization and automation. © 2019 The Authors. Basic Protocol 1: Tissue sampling methods and procedure Basic Protocol 2: Sample verification and DNA lysis Basic Protocol 3: Design of a genotyping strategy Basic Protocol 4: Moving to high-throughput genotyping.

Keywords: 3Rs; PCR; automation; genotyping; molecular biology; mouse model; replacement, reduction, refinement; reproducibility.

MeSH terms

  • Animals
  • Genotyping Techniques / instrumentation
  • Genotyping Techniques / methods*
  • High-Throughput Nucleotide Sequencing / instrumentation
  • High-Throughput Nucleotide Sequencing / methods*
  • Mice
  • Mutation*
  • Polymerase Chain Reaction / methods*
  • Reproducibility of Results