Open Access Thesis
Master of Science (M.S.)
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Month Degree Awarded
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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.
de Gannes, Matthew K., "Rapid Method of Processing Sperm for Nucleic Acid Extraction in Clinical Research" (2014). Masters Theses. 9.
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