TY  - GEN
T1  - A simple one-step PCR assay for SNP detection
AU  - Chen, Jian
AU  - Schedl, Tim
DO  - 10.17912/micropub.biology.000399
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000399/
AB  - Single nucleotide polymorphisms (SNPs) are the most common genetic variation between natural populations of a species, are the frequent cause of phenotypes observed in organisms from forward genetic chemical mutagenesis screens, and are employed to probe the functional consequences of missense and noncoding changes made by CRISPR/Cas9 gene editing (Brenner, 1974; Okamoto et al., 2019; Thompson et al., 2013; Zhao et al., 2014). For model organisms, SNP genotyping is essential for strain construction and is an important part of strain validation necessary for research rigor and reproducibility. This is particularly the case if the SNP does not have a known phenotype, does not have an easy way to score phenotype, or the phenotype is masked by epistasis. Additionally, laboratories are typically working with multiple genes, often examining multiple alleles, which could be legacy mutations or newly discovered/generated variants. Therefore, a simple SNP detection approach is desirable to allow rapid genotyping. Furthermore, the approach should be nimble in the sense of being applicable to any gene, potentially any SNP, and the generation of a new assay and its execution being straightforward and occurring in a short time frame. Here we describe a simple system for single worm genotyping. The strategy employs the differential efficiency of genomic PCR using a primer that has a single mismatch with the chromosome that contains the SNP to be detected (typically the variant allele) versus two mismatches with the corresponding alternative allele (typically the wild type allele). The genotype of a worm is deduced from two samples of the single worm lysis, where one PCR reaction uses a primer with mismatches to specifically detect the variant and not the wild type allele and the other PCR reaction uses a primer with mismatches to specifically detect the wild type allele and not the variant.
PY  - 2021
JO  - microPublication Biology
ER  -