![]() PCR can also fail to amplify certain templates, such as GC-rich sequences or secondary structures. Conversely, because of its low tolerance of inhibitors, frequent false negative results are also encountered (i.e., no amplification Bacich, Sobek, Cummings, Atwood, & O'Keefe, 2011 Schrader, Schielke, Ellerbroek, & Johne, 2012). Because of its high sensitivity, PCR is subject to frequent false positive results (i.e., amplification of a contaminant). Wrongly, PCR genotyping is often considered as a straightforward and easy step in reality, however, providing robust, accurate, and fast results is frequently more challenging that it seems. Many rodent genotyping protocols are based on polymerase chain reaction (PCR) amplification of genes or genetic markers, as PCR is easy, fast, sensitive, and cost effective. © 2019 The Authors.īasic Protocol 1: Tissue sampling methods and procedureīasic Protocol 2: Sample verification and DNA lysisīasic Protocol 3: Design of a genotyping strategyīasic Protocol 4: Moving to high-throughput 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. 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. Here we describe polymerase chain reaction (PCR) genotyping protocols for fast, sensitive, easy, and cost-effective characterization of mouse genotype. 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. ![]() Genotyping consists of searching for a DNA sequence variation localized at a well-defined locus in the genome.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |