Work

Rapid Method for the Isolation of Mammalian Sperm DNA

Year: 2015

Type: article

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 >90% high-quality DNA using 3 different commercially available silica-based spin columns. DNA yields did not differ between immediate isolation (2.84 ± 0.04 pg/cell) and isolation after 2 weeks of homogenate storage at room temperature (2.91 ± 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.

Source: BioTechniques

DOI: 10.2144/000114280

Authors: Haotian Wu, Matthew K. de Gannes, Gianna Luchetti, J.Richard Pilsner

Institution: University of Massachusetts Amherst

Language: English

Topic: Sperm and Testicular Function

Subfield: Reproductive Medicine

Field: Medicine

Domain: Health Sciences

Open Access status: gold

Estimated APC paid: $1,415

Funders: National Institutes of Health, National Institute of Environmental Health Sciences