{"title":"Alkaline dip DNA extraction from skin mucus for high-throughput sexing of sterlets (<i>Acipenser ruthenus</i>).","authors":"Ryuhei Kinami, Toshinao Ineno","doi":"10.1080/07366205.2025.2467584","DOIUrl":null,"url":null,"abstract":"<p><p>Studies on simple DNA extraction methods from fish mucus, which is an ideal resource for noninvasive sampling, are scarce. In the aquaculture of sturgeons such as sterlets (<i>Acipenser ruthenus</i>), a high-throughput genetic sexing method is needed, as only females are reared to maturity for their roe. Here, DNA extraction methods using HotSHOT (hot sodium hydroxide and tris), a novel alkaline dip, alkaline glycol, and water were compared using the skin mucus of sterlets (n = 8) collected with a toothpick. High-throughput sexing of sterlets from six production batches (n = 3953) was also evaluated using HotSHOT or alkaline dip. An alkaline dip with 10-25 mM NaOH was an effective alternative to HotSHOT, eliminating the heating and neutralizing steps. Regarding high-throughput sexing, > 99% (3946) individuals were successfully genotyped on the first PCR trial, and genotype ratios of each batch were close to 1:1, showing applicability of the alkaline dip method for practical sterlet sexing.</p>","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"1-10"},"PeriodicalIF":2.2000,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"BioTechniques","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1080/07366205.2025.2467584","RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
摘要
鱼类粘液是一种理想的非侵入性取样资源，但有关从鱼类粘液中提取 DNA 的简单方法的研究却很少。在养殖鲟鱼（如中华鲟）的过程中，需要一种高通量的基因性别鉴定方法，因为只有雌性鲟鱼才能饲养到成熟，以获得鱼子。在此，使用牙签采集的硬骨鱼（n = 8）的皮肤粘液，比较了使用 HotSHOT（热氢氧化钠和三羟甲基丙烷）、新型碱性浸液、碱性乙二醇和水提取 DNA 的方法。此外，还使用 HotSHOT 或碱性浸液对来自六个生产批次（n = 3953）的立体小鼠进行了高通量性别鉴定评估。用 10-25 mM NaOH 进行碱性浸泡可有效替代 HotSHOT，省去了加热和中和步骤。在高通量性别鉴定方面，在第一次 PCR 试验中，99% 以上（3946 个）的个体成功进行了基因分型，每批的基因型比率接近 1:1，这表明碱性浸渍法适用于实际的立体小鼠性别鉴定。
Alkaline dip DNA extraction from skin mucus for high-throughput sexing of sterlets (Acipenser ruthenus).
Studies on simple DNA extraction methods from fish mucus, which is an ideal resource for noninvasive sampling, are scarce. In the aquaculture of sturgeons such as sterlets (Acipenser ruthenus), a high-throughput genetic sexing method is needed, as only females are reared to maturity for their roe. Here, DNA extraction methods using HotSHOT (hot sodium hydroxide and tris), a novel alkaline dip, alkaline glycol, and water were compared using the skin mucus of sterlets (n = 8) collected with a toothpick. High-throughput sexing of sterlets from six production batches (n = 3953) was also evaluated using HotSHOT or alkaline dip. An alkaline dip with 10-25 mM NaOH was an effective alternative to HotSHOT, eliminating the heating and neutralizing steps. Regarding high-throughput sexing, > 99% (3946) individuals were successfully genotyped on the first PCR trial, and genotype ratios of each batch were close to 1:1, showing applicability of the alkaline dip method for practical sterlet sexing.
期刊介绍：
BioTechniques is a peer-reviewed, open-access journal dedicated to publishing original laboratory methods, related technical and software tools, and methods-oriented review articles that are of broad interest to professional life scientists, as well as to scientists from other disciplines (e.g., chemistry, physics, computer science, plant and agricultural science and climate science) interested in life science applications for their technologies.
Since 1983, BioTechniques has been a leading peer-reviewed journal for methods-related research. The journal considers:
Reports describing innovative new methods, platforms and software, substantive modifications to existing methods, or innovative applications of existing methods, techniques & tools to new models or scientific questions
Descriptions of technical tools that facilitate the design or performance of experiments or data analysis, such as software and simple laboratory devices
Surveys of technical approaches related to broad fields of research
Reviews discussing advancements in techniques and methods related to broad fields of research
Letters to the Editor and Expert Opinions highlighting interesting observations or cautionary tales concerning experimental design, methodology or analysis.
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