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首页> 外文期刊>Journal of Experimental Marine Biology and Ecology >An effective procedure for DNA isolation and voucher recovery from millimeter-scale copepods and new primers for the 18S rRNA and cytb genes
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An effective procedure for DNA isolation and voucher recovery from millimeter-scale copepods and new primers for the 18S rRNA and cytb genes

机译：从毫米级co足类动物和18S rRNA和cytb基因的新引物分离DNA和回收凭单的有效程序

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摘要

Many investigators need to determine whether individuals belong to the same species. DNA-sequence data have helped with this task, but current procedures of DNA isolation from millimeter-scale crustaceans, such as harpacticoid copepods, leave little to no voucher material for morphological analysis, and many procedures yield only enough DNA for a single amplification reaction. We therefore developed a DNA-isolation procedure that yielded essentially intactexoskeletons and sufficient DNA for multiple polymerase chain reactions. DNA-amplification success of our DNA-isolation procedure was relatively insensitive to (1) the length of preservation time from sample collection to DNA isolations and (2) the length of time the DNA was stored at -20 ℃ after isolation. An additional benefit of our procedure is therefore that the DNA isolated is relatively stable. Primers available for the nuclear 18S rRNA gene and the mitochondrial cytochrome oxidase b (cytb) gene are known not to work for many harpacticoids. We therefore designed primers that would amplify and sequence an ～750-base-pair fragment of the 18S rRNA gene and others that would amplify and sequence an ～450-base-pair fragment of the cytb gene. Both primer sets worked for at least 12 harpacticoid families.
机译：许多研究人员需要确定个人是否属于同一物种。 DNA序列数据帮助完成了这一任务，但是当前从毫米级甲壳类动物中分离DNA的程序（例如类拟actic足类足纲动物）几乎没有凭证材料用于形态分析，而且许多程序只能产生足够的DNA来进行单个扩增反应。因此，我们开发了一种DNA分离程序，该程序可产生基本上完整的外骨骼和足够的DNA用于多种聚合酶链反应。我们的DNA分离方法的DNA扩增成功对（1）从样品收集到DNA分离的保存时间长短以及（2）分离后在-20℃保存DNA的时间长短相对不敏感。因此，我们程序的另一个好处是分离出的DNA相对稳定。已知可用于核18S rRNA基因和线粒体细胞色素氧化酶b（cytb）基因的引物不适用于许多类类拟兽药。因此，我们设计了能扩增和测序18S rRNA基因〜750个碱基对片段的引物，以及其他能扩增cytb基因〜450个碱基对片段的引物。两种引物对至少12个类类拟螺齿科均起作用。

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