Background: Sperm contain highly compact nuclei, inhibiting DNA extraction using traditional techniques. Current methods extracting sperm DNA involve lengthy lysis and no means of stabilizing DNA, hindering clinical research.
Objective: We sought to optimize an efficient method of extracting high quality human sperm DNA.
Methods: Sperm from three volunteers were isolated using PureCeption. We tested 1) proteinase K with DNA/RNA Shield, 2) DTT and TCEP as reducing agents, 3) QIAshredder homogenization, and 4) stability of sperm DNA fresh (baseline) or after 4 weeks of storage at 4OC in DNA/RNA Shield using modified Quick-gDNA MiniPrep. DNA was PCR amplified using ALU primers and digested with Hinf1 restriction enzymes. DNA methylation was measured by MassARRAY.
Results: DNA concentrations were similar with (30.1+0.28ng/μL, 33.4+0.21ng/μL) and without (28.9+0.00ng/μL, 30.9+0.85ng/μL) proteinase K. Sperm cells were lysed after 1 and 20 minutes with 25mM TCEP and 100mM DTT respectively. TCEP (50mM) produced greater DNA concentrations (17.2+0.50ng/μL, 21.3+0.71ng/μL) than 50mM DTT (12.6+0.28ng/μL, 12.3+0.35ng/μL). Adding QIAshredder to 50mM TCEP increased DNA concentrations (25.9+0.35ng/μL, 21.7+0.49ng/μL versus 18.6+0.99ng/μL, 12.3+0.35ng/μL). At baseline and 4 weeks: 1) DNA concentrations were similar (36.2+2.75 ng/μL, 32.2+1.38ng/μL, 44.3+3.93ng/μL versus 40.0+2.98ng/μL, 37.6+1.38ng/μL, 38.7+3.93ng/μL respectively) and 2) DNA was equally amplified by PCR and digested with restriction enzymes. DNA methylation was similar at baseline and 4 weeks for SNURF (1.43+1.02%, 1.55+0.95%), PEG10 (3.69+0.66%, 4.28+1.52%), and H19 (88.93+3.24%, 91.78+2.00%).
Conclusions: We stabilized and isolated high quality DNA from human sperm using 5 minute versus > 2 hour lysis in other methods. Our methods may facilitate efficient clinical research.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:masters_theses_2-1020 |
| Date | 29 August 2014 |
| Creators | de Gannes, Matthew K |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses |
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