AgriBio Centre for AgriBioscience, Department of Animal, Plant and Soil Sciences, School of Life Sciences, La Trobe University, 5 Ring Road, Bundoora, Victoria, 3086, Australia.
School of Science, Psychology and Sport, Federation University, Churchill, Victoria, Australia.
BMC Biotechnol. 2021 May 17;21(1):35. doi: 10.1186/s12896-021-00695-6.
The purpose of this study was to develop a reliable DNA extraction protocol to use on individual Teladorsagia circumcincta nematode specimens to produce high quality DNA for genome sequencing and phylogenetic analysis. Pooled samples have been critical in providing the groundwork for T. circumcincta genome construction, but there is currently no standard method for extracting high-quality DNA from individual nematodes. 11 extraction kits were compared based on DNA quality, yield, and processing time.
11 extraction protocols were compared, and the concentration and purity of the extracted DNA was quantified. Median DNA concentration among all methods measured on NanoDrop 2000™ ranged between 0.45-11.5 ng/μL, and on Qubit™ ranged between undetectable - 0.962 ng/μL. Median A260/280 ranged between 0.505-3.925, and median A260/230 ranged - 0.005 - 1.545. Larval exsheathment to remove the nematode cuticle negatively impacted DNA concentration and purity.
A Schistosoma sp. DNA extraction method was determined as most suitable for individual T. circumcincta nematode specimens due to its resulting DNA concentration, purity, and relatively fast processing time.
本研究的目的是开发一种可靠的 DNA 提取方案，用于单个圆形阔节毛线虫线虫标本，以产生高质量的 DNA 用于基因组测序和系统发育分析。混合样本在提供圆形阔节毛线虫基因组构建的基础方面至关重要，但目前尚无从单个线虫中提取高质量 DNA 的标准方法。本研究基于 DNA 质量、产量和处理时间比较了 11 种提取试剂盒。
比较了 11 种提取方案，并对提取 DNA 的浓度和纯度进行了定量。所有方法在 NanoDrop 2000™上测量的 DNA 浓度中位数在 0.45-11.5ng/μL 之间，在 Qubit™上测量的 DNA 浓度中位数在不可检测到 0.962ng/μL 之间。A260/280 的中位数在 0.505-3.925 之间，A260/230 的中位数在-0.005-1.545 之间。幼虫蜕皮以去除线虫角质层会对 DNA 浓度和纯度产生负面影响。
由于其产生的 DNA 浓度、纯度和相对较快的处理时间，确定旋毛虫 DNA 提取方法最适合单个圆形阔节毛线虫线虫标本。
