IMP PCR primers detect single nucleotide polymorphisms for Anopheles gambiae species identification, Mopti and Savanna rDNA types, and resistance to dieldrin in Anopheles arabiensis
IMP PCR primers detect single nucleotide polymorphisms for Anopheles gambiae species identification, Mopti and Savanna rDNA types, and resistance to dieldrin in Anopheles arabiensis
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DOI：
10.1186/1475-2875-5-125
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发表时间：
2006-12-19
期刊：
影响因子：
3
通讯作者：
Benedict, Mark Q.
中科院分区：
文献类型：
--
作者：
Wilkins, Elien E.;Howell, Paul I.;Benedict, Mark Q.
背景：区分单核苷酸多态性的聚合酶链反应通常用于蚊子鉴定和杀虫剂抗性等位基因的鉴定。然而，用于引物设计的现有方法通常会导致分析不够稳健或需要额外的步骤。 方法：利用与两个模板的 3 引物末端 SNP 三个碱基故意错配的独特寡核苷酸，开发并测试了三种新的 PCR 测定法，这些测定法使用未消化 DNA 片段的标准凝胶电泳来区分 SNP 靶标。这些应用于：（1）另一种核糖体 DNA PCR 测定法，用于区分冈比亚按蚊复合体的五个成员； (2)莫普提和萨凡纳rDNA类型的检测； (3) 区分阿拉伯按蚊对狄氏剂 (Rdl) 等位基因抗性的测定。结果：在所有三种测定中均观察到目标等位基因的可重复特异性扩增。结果与现有分析一致，但事实证明更简单，结果在我们手中更清晰。结论：该方法的简单性和有效性应在这些和其他 PCR 分析中利用，以提高其特异性和简单性。这些结果不仅有可能扩展到蚊子分析，还可以扩展到寄生虫和人类多态性。
Background: Polymerase chain reactions to distinguish single-nucleotide polymorphisms are commonly used for mosquito identification and identifying insecticide resistance alleles. However, the existing methods used for primer design often result in analyses that are not robust or require additional steps.Methods: Utilizing oligonucleotides that are unique in having an intentional mismatch to both templates three bases from the SNP at the 3-prime end, three new PCR assays that distinguish SNP targets using standard gel electrophoresis of undigested DNA fragments were developed and tested. These were applied to: ( 1) an alternative ribosomal DNA PCR assay to distinguish five members of the Anopheles gambiae complex; ( 2) detection of the Mopti and Savanna rDNA types; and ( 3) an assay to distinguish resistance to dieldrin (Rdl) alleles in Anopheles arabiensis.Results: Reproducible specific amplification of the target alleles was observed in all three assays. The results were consistent with existing analyses but proved simpler and the results more distinct in our hands.Conclusion: The simplicity and effectiveness of the method should be utilized in these and other PCR analyses to increase their specificity and simplicity. These results have the potential to be extended not only to mosquito analyses but also to parasite and human polymorphisms.