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. 2015 Jun 1;58(6):293-300.
doi: 10.2144/000114280. eCollection 2015 Jun.

Rapid method for the isolation of mammalian sperm DNA

Haotian Wu  1 , Matthew K de Gannes  1 , Gianna Luchetti  1 , J Richard Pilsner  1
Affiliations

Rapid method for the isolation of mammalian sperm DNA

Haotian Wu et al. Biotechniques. .

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.

Keywords: DNA isolation; DNA purification; epigenetics; genetics; methods; nucleic acids; sperm; spermatozoa.

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Figures

Figure 1
Figure 1. Utility of tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent for the isolation of sperm DNA
(A) Mean (±SD) DNA yield (pg/cell) of sperm cells treated with Buffer RLT, either 50 mM TCEP or 150 mM DTT, and proteinase K (ProK) for two hours at 56°C. DNA was isolated from sperm cell lysate via AllPrep DNA columns. (B) Mean (±SD) DNA yield (pg/cell) of sperm cells treated with Buffer RLT (50 mM TCEP) and the following homogenization methods: none; Qiashredder (as directed); or 0.1 g of 0.2 mm stainless steel beads. DNA was isolated from sperm cell lysate via AllPrep DNA columns. (C) Mean (±SD) DNA yield (pg/cell) of sperm cells purified with Qiagen AllPrep, QIAmp Mini, or Zymo Quick-gDNA spin columns. Sperm cells were homogenized by 0.2 mm stainless steel beads in either Buffer RLT (AllPrep and DNA Mini) or Zymo gDNA lysis buffer (Quick gDNA), both supplemented with 50 mM TCEP. (D) Mean (±SD) DNA yield (pg/cell) of sperm cells homogenized by 0.2 mm stainless steel beads in the presence of Buffer RLT and 50 mM TCEP. DNA was isolated from sperm cell lysate via AllPrep spin columns processed immediately (T0) and after 2 weeks storage (T2) at room temperature (22°C).
Figure 2
Figure 2. Electrophoresis of sperm DNA
Sperm DNA isolated from 3 individuals immediately (T0) and after 2 weeks of storage at room temperature (T2) on a 0.7% agarose gel.
Figure 3
Figure 3. DNA methylation of three imprinted loci
Mean (±SD) percentage DNA methylation of CpG sites within imprinted regions of SNURF, PEG10, and H19 using DNA isolated from sperm lysate immediately (T0) and after 2 weeks of storage at room temperature (T2).
Figure 4
Figure 4. Schematic of sperm DNA isolation workflow
Sperm cells are isolated from ejaculate via gradient centrifugation with 90% PureCeption. Pelleted sperm cells are then homogenized with Buffer RLT, 50 mM tris(2-carboxyethyl)phosphine (TCEP), and 0.1 g of 0.2 mm stainless steel beads on a Disruptor Genie for 5 min. This produces a working lysate that is ready for DNA and RNA isolation.
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