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A Rapid, Super-Selective Method for Detection of Single Nucleotide Variants in Caenorhabditis elegans.
GENETICS ( IF 3.3 ) Pub Date : 2020-08-17 , DOI: 10.1534/genetics.120.303553
Denis Touroutine 1 , Jessica E Tanis 2
Affiliation  

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 Caenorhabditiselegans Each SS primer consists of a 5' "anchor" that hybridizes to the template, followed by a non-complementary "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.

中文翻译:

一种快速、超选择性检测秀丽隐杆线虫单核苷酸变异的方法。

随着通过诱变筛选和基因组编辑技术产生的单核苷酸变体的广泛使用,迫切需要一种有效且低成本的策略来对单核苷酸取代进行基因分型。我们开发了一种快速且廉价的方法,通过优化用于秀丽隐杆线虫终点 PCR 的超选择性 (SS) 引物来检测点突变体。每个SS 引物由与模板杂交的 5'“锚”组成,后面是非-互补的“桥”和对应于目标等位基因的“脚”。脚序列很短,因此末端 3' 核苷酸处的单个错配会破坏引物结合的稳定性并阻止延伸,从而能够区分不同的等位基因。我们探索了每个 SS 引物片段的长度和序列组成如何影响各种遗传环境中的选择性和效率,以便制定简单的引物设计规则,允许在广泛的退火温度下区分等位基因。操纵桥长度会影响放大效率,而修改脚序列会改变辨别力。改变锚定位置使得 SS 引物能够用于对标准引物设计具有挑战性的序列区域进行基因分型。在定义引物设计参数后,我们证明了 SS 引物对粗线虫裂解物进行基因分型的实用性,这表明该方法也可用于 SNP 作图和 CRISPR 突变体的筛选。此外,由于 SS 引物能够可靠地检测点突变,因此该方法具有在所有遗传系统中广泛应用的潜力。
更新日期:2020-08-24
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