High quality genomic DNA extraction from large milk samples

Abstract

K: Cattle, milk, DNA, SCC, PCR, EDTA.Recently, techniques have been reported which describe the isolation of genomic DNA from somatic cells of bovine milk (Lipkin et al. 1993(Lipkin et al. , 1998, epithelial cells of human milk (Lindquist et al. 1994) and bovine and caprine milk (Amills et al. 1997). In each of these reports, somatic cell DNA was successfully amplified by polymerase chain reaction (PCR). However, none of these methods are suitable for large-scale genotyping projects, because consistent quantifiable amounts of good quality genomic DNA were not obtained from milk somatic cells, or because good quality PCR product could not consistently be amplified from genomic DNA.The somatic cell count (SCC) in bovine milk increases in response to infection from a low range between 2i10% and 2i10& cells\ml from a healthy quarter, to a high range from 3i10& to 1i10( cells\ml from an infected quarter (Holmes & Wilson, 1984 ; Kehrli & Shuster, 1994). In whole blood, the numbers of white blood cells (nucleated cells which can yield genomic DNA) also vary in response to infection, ranging between 7i10' and 1i10 ( cells\ml (Swenson, 1993). Whole blood can contain up to 350 times more nucleated cells than milk, when comparing lower cell counts for the same volumes of whole blood and milk.A volume of 500 µl whole blood can typically be used to provide adequate genomic DNA for PCR-based techniques. Conservatively, 500 µl whole blood should yield approximately 3n5i10' copies of genomic DNA, which is equivalent to 11n7 µg mammalian genomic DNA (Innis & Gelfand, 1990). A 50-ml volume of milk from an uninfected quarter with a very low SCC should yield about 3n3 µg genomic DNA, providing the technique for isolating genomic DNA is of similar efficiency.In a preliminary report (Murphy et al. 1995), we demonstrated that good quality genomic DNA could be isolated from 1n5-ml samples of milk collected from single quarters. However, this method was not reliable for isolating DNA for large-scale genotyping projects, because of the highly variable yields of genomic DNA. If DNA was isolated directly from 50 ml milk, then the resulting genomic DNA was severely degraded because casein interfered with Proteinase K digestion of enzymes that degrade somatic cell DNA. Good quality genomic DNA could be isolated from 50-ml samples only if the somatic cells were separated from casein prior to Proteinase K digestion. Previously, this involved laborious procedures of somatic cell aspiration and although the final yield of genomic DNA was of high quality, it was also highly variable in quantity. In the current report we demonstrate radical changes to the

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

“…The first modification was the addition of a TE-EDTA solution to dissolve milk casein. The latter is an undesirable substance for DNA extraction because it interferes with the digestion by proteinase K, of cellular enzymes which are responsible for degradation of the DNA [8]. Milk casein contains calcium and EDTA chelates calcium.…”

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

“…In early studies, the phenol-chloroform method was generally used for DNA extraction from different kind of tissues, but it was not applicable for largescale studies because sufficient amounts of good quality DNA were not obtained from milk samples (Lipkin et al, 1993;Murphy et al, 1995). Recently, several studies have reported that milk somatic cells can be used as a source of DNA (Murphy et al, 2002;Mihuaiu et al, 2009;Psifidi et al, 2010), but the high quantity and quality genomic DNA isolation from milk is still a major concern. The present study demonstrated that compared to the classical phenolchloroform method, the Modified Nucleospin Blood kit method was a bit expensive but was found to be efficient and the best method to obtain highly intact DNA of improved quantity and purity, and that the Modified TianGen kit method was next to the Modified Nucleospin Blood kit method in terms of DNA quantity and quality and lower cost.…”

“…In cattle, genomic DNA is normally extracted from peripheral blood leukocytes, different tissues or hair follicles. Although milk is animal friendly and a routine source in the dairy industry (D'Angelo et al, 2007), inhibitors in milk such as fats and proteins render it difficult source for extracting high quantity and quality DNA (Lipkin et al, 1993;Amills et al, 1997;Murphy et al, 2002;Feligini et al, 2005;Cremonesi et al, 2006). To date, different commercial kits for improved DNA extraction are available for many kinds of tissues except for milk (Biase et al, 2002;Studer et al, 2008;O'Grady et al, 2008;Gao et al, 2011).…”

Yield and amplificability of different DNA extraction procedures for traceability in the dairy food chain

“…Total DNA was extracted with four different commercial kits: NucleoSpin Ò Food (''Food") (Macherey-Nagel, Düren, Germany), Charge Switch Ò Forensic DNA Purification Kit (''Forensic") (Invitrogen), Wizard Ò Resin (''Wizard") (Promega, Madison, WI, USA), and QIAamp DNA Stool Ò Mini Kit (''Stool") (Qiagen, Hilden, Germany). Other protocols obtained from literature were tested: Tween-based method (''Tween") (Murphy, Shariflou, & Moran, 2002) and CTABbased method (''CTAB") (Nemeth et al, 2004). The CTAB-based method has been modified in our laboratory as follows: 300 mg of sample were incubated overnight at 60°C under agitation with 4.5 lL of Proteinase K (20 mg mL À1 ) and 900 lL of CTAB buffer (1.4 M NaCl, 2% (w/v) CTAB (hexadecyltrimethylammonium bromide), 100 mM Tris, 15 mM EDTA, pH 8.0).…”