Novel extraction method of genomic DNA suitable for long-fragment amplification from small amounts of milk

Abstract

Isolation of genomic DNA is a prerequisite for assessment of milk quality. As a source of genomic DNA, milk somatic cells from milking ruminants are practical, animal friendly, and cost-effective sources. Extracting DNA from milk can avoid the stress response caused by blood and tissue sampling of cows. In this study, we optimized a novel DNA extraction method for amplifying long (>1,000 bp) DNA fragments and used it to evaluate the isolation of DNA from small amounts of milk. The techniques used for the separation of milk somatic cell were explored and combined with a sodium dodecyl sulfate (SDS)-phenol method for optimizing DNA extraction from milk. Spectrophotometry was used to determine the concentration and purity of the extracted DNA. Gel electrophoresis and DNA amplification technologies were used for to determine DNA size and quality. The DNA of 112 cows was obtained from milk (samples of 13 ± 1 mL) and the corresponding optical density ratios at 260:280 nm were between 1.65 and 1.75. Concentrations were between 12 and 45 μg/μL and DNA size and quality were acceptable. The specific PCR amplification of 1,019- and 729-bp bovine DNA fragments was successfully carried out. This novel method can be used as a practical, fast, and economical mean for long genomic DNA extraction from a small amount of milk.

Changes in physicochemical properties and DNA quality of milk as affected by different heat treatments

“…Theoretically, pure DNA solutions have an A 260 /A 280 of above 1.8, but we found that the ratio of absorbance (A 260 /A 280 ) of all DNA solutions was below 1.8, probably due to contamination by phenols or proteins. For the raw milk, the resulting ratio (1.60) was lower than that (1.79) reported by Pokorska et al (2016), but similar to that (1.65) reported by Liu et al (2014). These differences in purity of the raw milk samples might be attributed to the different extraction method we used.…”

“…(), but similar to that (1.65) reported by Liu et al . (). These differences in purity of the raw milk samples might be attributed to the different extraction method we used.…”

“…Total DNA was isolated from 10 mL milk according to the method described by Liu et al (2014). The DNA solutions obtained from the samples of each treatment were used to evaluate the yield, purity and integrity of the DNA, for both mtDNA and nDNA.…”

Assessment of milk quality during storage based on DNA extracted from milk

“…The DNA was extracted from 10-mL milk according to the method described in our previous work (Liu et al, 2014). The method of DNA extraction could avoid a stress response in the sampled animals affecting milk production compared with traditional extraction methods, which obtained DNA from blood and organ tissues (Buitkamp & Gotz, 2004).…”

“…Compared to biochemical analysis, traditional sensory, chemical and instrumental methods used to assess milk quality are limited in analyzing large-scale samples (Guy & Fenaille, 2006;Korel & Balaban, 2002;Larsen, Kidmose, Kristensen, Beaumont, & Mortensen, 2013;Mariaca & Bosset, 1997;Marsili, 2000). In recent years, molecular techniques have been one of the powerful tools in quality inspection of dairy products and in research of molecular nutrition (Liu, Gao, Yang, Ku, & Zan, 2014). Whether this method can be utilized to monitor the milk quality remains unknown.…”