六种适用于基于 PCR 的基因分型应用的羊乳样本基因组 DNA 提取方法的比较。

A comparison of six methods for genomic DNA extraction suitable for PCR-based genotyping applications using ovine milk samples.

机构信息

Animal Production Laboratory, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece.

出版信息

Mol Cell Probes. 2010 Apr;24(2):93-8. doi: 10.1016/j.mcp.2009.11.001. Epub 2009 Nov 10.

DOI:10.1016/j.mcp.2009.11.001

PMID:19900535

Abstract

Isolation of amplifiable genomic DNA is a prerequisite for the genetic assessment of diseases and disease susceptibility in farm animals. Milk somatic cells are a practical, animal friendly and cost-effective source of genomic DNA in milking ruminants. In this study, six different DNA extraction methods were optimized, evaluated and compared for the isolation of DNA from ovine milk samples. Methods 1 and 2 were direct applications of two commercial kits, Nucleospin((R)) Blood and Nucleospin((R)) Tissue, respectively. Methods 3 and 4 were based on modified protocols of methods 1 and 2, respectively, aiming at increasing DNA recovery and integrity, and eliminating PCR inhibitors. Method 5 was a standard Phenol-Chloroform protocol application and method 6 was based on an in-house developed protocol using silica as the affinity matrix. Spectrophotometer, gel electrophoresis and real-time PCR measurements were used as criteria for evaluating quantity and quality of the extracted DNA. Processing time, intensity of labor and cost for each method were also evaluated. Results suggested that methods 1-4 were considered suitable for molecular downstream applications and performed better than methods 5 and 6. Modifications of protocols 3 and 4 increased the quantity and quality of the extracted DNA from ovine milk samples. Method 3 was proved to be highly efficient and robust for large scale use as demonstrated by its successful application to 1000 individual ovine milk and 50 bulk milk samples.

摘要

从奶样中分离可扩增的基因组 DNA 是对反刍动物疾病和疾病易感性进行遗传评估的前提。牛奶体细胞是从泌乳反刍动物奶样中提取基因组 DNA 的实用、对动物友好且具有成本效益的来源。本研究对 6 种不同的 DNA 提取方法进行了优化、评估和比较，以从绵羊奶样中分离 DNA。方法 1 和 2 分别是商业试剂盒 Nucleospin((R)) Blood 和 Nucleospin((R)) Tissue 的直接应用。方法 3 和 4 分别基于方法 1 和 2 的改良方案，旨在提高 DNA 的回收率和完整性，并消除 PCR 抑制剂。方法 5 是标准的酚氯仿法应用，方法 6 是基于使用硅胶作为亲和基质的内部开发方法。分光光度计、凝胶电泳和实时 PCR 测量被用作评估提取 DNA 的数量和质量的标准。还评估了每种方法的处理时间、劳动强度和成本。结果表明，方法 1-4 被认为适用于分子下游应用，且比方法 5 和 6 表现更好。方案 3 和 4 的改良提高了从绵羊奶样中提取的 DNA 的数量和质量。方法 3 被证明具有高效和稳健的特点，可大规模应用，其已成功应用于 1000 个个体绵羊奶和 50 个混合奶样。