A Rapid, SuperSelective Method for Detection of Single Nucleotide Variants in Caenorhabditis elegans

生物 底漆(化妆品) 遗传学 基因分型 秀丽隐杆线虫 点突变 单核苷酸多态性 放大器 SNP基因分型 计算生物学 基因型 突变体 聚合酶链反应 基因 化学 有机化学
作者
Denis Touroutine,Jessica E. Tanis
出处
期刊:Genetics [Oxford University Press]
卷期号:216 (2): 343-352 被引量:13
标识
DOI:10.1534/genetics.120.303553
摘要

Abstract With the widespread use of single nucleotide variants generated through mutagenesis screens and genome editing technologies, there is pressing need for an efficient and low-cost strategy to genotype single nucleotide substitutions. We have developed a rapid and inexpensive method for detection of point mutants through optimization of SuperSelective (SS) primers for end-point PCR in Caenorhabditis elegans. Each SS primer consists of a 5′ “anchor” that hybridizes to the template, followed by a noncomplementary “bridge,” and a “foot” corresponding to the target allele. The foot sequence is short, such that a single mismatch at the terminal 3′ nucleotide destabilizes primer binding and prevents extension, enabling discrimination of different alleles. We explored how length and sequence composition of each SS primer segment affected selectivity and efficiency in various genetic contexts in order to develop simple rules for primer design that allow for differentiation between alleles over a broad range of annealing temperatures. Manipulating bridge length affected amplification efficiency, while modifying the foot sequence altered discriminatory power. Changing the anchor position enabled SS primers to be used for genotyping in regions with sequences that are challenging for standard primer design. After defining primer design parameters, we demonstrated the utility of SS primers for genotyping crude C. elegans lysates, suggesting that this approach could also be used for SNP mapping and screening of CRISPR mutants. Further, since SS primers reliably detect point mutations, this method has potential for broad application in all genetic systems.

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