title: Rapid method for the isolation of mammalian sperm DNA.
abstract: The unique DNA packaging of spermatozoa renders them resistant to DNA isolation techniques used for somatic cells, requiring alternative methods that are slow and labor intensive. Here we present a rapid method for isolating high-quality sperm DNA. Isolated human sperm cells were homogenized with 0.2 mm steel beads for 5 min at room temperature in the presence of guanidine thiocyanate lysis buffer supplemented with 50 mM tris(2-carboxyethyl)phosphine (TCEP). Our method yielded &gt;90% high-quality DNA using 3 different commercially available silica-based spin columns. DNA yields did not differ between immediate isolation (2.84 &plusmn; 0.04 pg/cell) and isolation after 2 weeks of homogenate storage at room temperature (2.91 &plusmn; 0.13 pg/cell). DNA methylation analyses revealed similar methylation levels at both time points for three imprinted loci. Our protocol has many advantages: it is conducted at room temperature; lengthy proteinase K (ProK) digestions are eliminated; the reducing agent, TCEP, is odorless and stable at room temperature; nucleic acids are stabilized, allowing storage of homogenate; and it is adaptable for other mammalian species. Taken together, the benefits of our improved method have important implications for settings where sample processing constraints exist.
authors.affiliation: Department of Environmental Health Sciences, School of Public Health and Health Sciences, University of Massachusetts Amherst, Amherst, MA.
authors.forename: Haotian
authors.initials: H
authors.lastname: Wu
authors.affiliation: Department of Environmental Health Sciences, School of Public Health and Health Sciences, University of Massachusetts Amherst, Amherst, MA.
authors.forename: Matthew K
authors.initials: MK
authors.lastname: de Gannes
authors.affiliation: Department of Environmental Health Sciences, School of Public Health and Health Sciences, University of Massachusetts Amherst, Amherst, MA.
authors.forename: Gianna
authors.initials: G
authors.lastname: Luchetti
authors.affiliation: Department of Environmental Health Sciences, School of Public Health and Health Sciences, University of Massachusetts Amherst, Amherst, MA.
authors.forename: J Richard
authors.initials: JR
authors.lastname: Pilsner
mesh_terms: D000818:Animals; D002469:Cell Separation; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D019175:DNA Methylation; D004586:Electrophoresis / Q000379:methods; D006801:Humans; D008297:Male; D010720:Phosphines / Q000737:chemistry; D019163:Reducing Agents / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D013094:Spermatozoa / Q000737:chemistry*
chemical_list: D004247:DNA; D010720:Phosphines; D019163:Reducing Agents; D012822:Silicon Dioxide; C080938:tris(2-carboxyethyl)phosphine
keywords: DNA isolation; DNA purification; epigenetics; genetics; methods; nucleic acids; sperm; spermatozoa