Comparison of methods for high quantity and quality genomic DNA extraction from raw cow milk

Abstract

ABSTRACT. Isolation of sufficient quantities of high quality DNA is a prerequisite for molecular studies. Milk somatic cells can be used; however, inhibitors such as fats and proteins make milk a difficult medium for extracting large amounts of quality DNA. We optimized, evaluated and compared three methods, Modified Nucleospin Blood Kit method, Modified TianGen Kit method and Phenol-Chloroform method for genomic DNA extraction from bovine milk. Individual cows' milk and bulk milk samples were collected from a China agricultural university dairy farm. Genomic DNA extracted from each milk sample by the three methods was evaluated for quantity and purity by spectrophotometry and gel electrophoresis, as well as PCR and sequencing. All the three methods were found suitable for genomic DNA isolation from bovine milk, PCR applications, and sequencing. Comparing the three methods, we found that the Modified Nucleospin Blood Kit method was significantly better than the Phenol-Chloroform method in terms of quantity as well as quality (amount, concentration, 260/280nm and 260/230nm absorbance ratio), whereas, the Modified TianGen Kit method was more efficient than the Phenol-Chloroform method and cheaper than the Modified Nucleospine Blood Kit method; it yielded reasonably good quantities of good quality DNA and would be suitable for large-scale genotyping of lactating cows.

Gap analysis ofMycoplasma bovisdisease, diagnosis and control: An aid to identify future development requirements

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New Rapid Method of DNA Isolation from Milk Somatic Cells

“…Although the concentration of somatic cells in 1 mL of milk (usually 2 × 10 4 to 4 × 10 5 ) is much lower than the concentration of leukocytes in 1 mL of blood (usually 4 × 10 6 to 10 × 10 6 ), and despite the fact that milk contains inhibitors such as fat and protein, the isolation of DNA from milk is feasible ( 13 14 15 16 ). Hitherto known methods of DNA extraction from milk somatic cells are often time consuming, expensive, and require a relatively large volume of milk (15–50 mL) and use of toxic reagents ( 14 15 16 17 18 19 20 ). Bearing in mind the aforementioned, a new, fast, nontoxic, and inexpensive method of DNA isolation from small amounts of raw milk was developed at the Department of Cattle Breeding at the University of Agriculture in Krakow (patent application number: P.404 447 in Poland).…”

Development of a Membrane-Based Method for Isolation of Genomic DNA from Human Blood

“…Extraction of high-quality, intact, double-stranded, highquantity, protein, and PCR inhibitor-free gDNA is a primary requisite for successful and reproducible genomic analysis and other molecular biology applications. 12 Several organic extraction methods, 13 nonorganic extraction methods, 13 and spin column-based extraction methods 2,14,15 are known and used extensively. The use of these methods is, however, limited by the following: 1) use of toxic and/or expensive reagents, 2) being laborious, 3) the requirement of large numbers of steps, 4) extended hands-on time, and 5) cost, among others.…”