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High-throughput DNA extraction for PCR-based genotyping of single Penaeus monodon embryos and nauplii
2010 Published by ElsevierB.V.
【Abstract】: A simple technique is described for isolating DNA from individual embryos or nauplii of the Black Tiger shrimp, Penaeus monodon. The protocol used a commercial prepGEM™ insect DNA extraction kit in combination with water bath ultrasonic disruption. DNA quality and yields were sufficient for multiplex PCR discrimination of up to six DNA microsatellites in a single test. The utility of the method was demonstrated in defining the parentage of 1279 embryos (7 h old) spawned from 21 females and 684 newly-hatched N1 to N2 nauplii spawned from 12 females each inseminated artificially with the spermatophores of two different males. Overall, parentage assignments based on the six microsatellite markers were deduced to be 89.5±6.7% and 93.0±4.4% effective using DNA isolated from individual embryos and nauplii, respectively. The technique is simple, cost-effective and should provide a robust means of isolating DNA from individual embryos and nauplii of aquatic invertebrates suitable for PCR-based genotyping.
【摘要】：本文描述了一种从斑节对虾、黑虎虾的胚胎或幼体个体提取DNA的简单方法，主要是用商业化的prepGEM™昆虫提取试剂盒，同时结合水浴超声破碎。DNA质量及产量足够用于一次反应中多达6对微卫星DNA的多重PCR检测。此方法以已经在1279胚胎（7小时生命）亲子鉴定的实验中证实，这些胚胎来自21个雌体及各与2个雄体经过人工受精后的12个雌体新孵出的684个N1-N2幼体。基于胚胎或幼体个体DNA进行的6对微卫星标记的亲系鉴定，推导为89.5±6.7%和93.0±4.4%。此技术简单高效，节约成本，为水生无脊椎动物胚胎和幼体PCR基因分型提供了极佳的的DNA提取方法。