Department of Entomology, Purdue University, Smith Hall, 901 West State Street, West Lafayette, IN 47907, USA.
Mol Biol Rep. 2010 Oct;37(7):3631-5. doi: 10.1007/s11033-010-0014-5. Epub 2010 Mar 4.
Genomic DNA extraction protocols generally require the use of expensive and hazardous reagents necessary for decontamination of phenolic compounds from the extracts. In addition, they are lengthy, hindering large-scale sample extractions necessary for high-throughput analyses. Here we describe a simple, time and cost-efficient method for genomic DNA extraction from insects. The extracted DNA was successfully used in a Polymerase Chain Reaction (PCR), making it suitable for automation for large-scale genetic analysis and barcoding studies. The protocol employs a single purification step to remove polysaccharides and other contaminating compounds using a non-hazardous reagent buffer. In addition, we conducted a bioinformatics database analysis as proof of concept for the efficiency of the DNA extraction protocol by using universal barcoding primers specific for cytochrome c oxidase I gene to identify different arthropod specimens through Barcode of Life Database (BOLD) database search. The usefulness of this protocol in various molecular biology and biodiversity studies is further discussed.
基因组 DNA 提取方案通常需要使用昂贵且危险的试剂，这些试剂用于去除提取物中的酚类化合物。此外，这些方案还很冗长，妨碍了高通量分析所需的大规模样本提取。在这里，我们描述了一种从昆虫中提取基因组 DNA 的简单、省时、高效的方法。提取的 DNA 成功地用于聚合酶链反应（PCR），使其适合于大规模遗传分析和条形码研究的自动化。该方案仅使用单一的纯化步骤，使用非危险的试剂缓冲液去除多糖和其他污染物。此外，我们还进行了生物信息学数据库分析，通过使用针对细胞色素 c 氧化酶 I 基因的通用条形码引物，通过条形码生命数据库（BOLD）数据库搜索来鉴定不同的节肢动物标本，以此作为该 DNA 提取方案效率的概念验证。此外，还进一步讨论了该方案在各种分子生物学和生物多样性研究中的用途。
