Ihsan Nalkiran, Levent Tumkaya, Sema Yilmaz Rakici, Hatice Sevim Nalkiran, Ali Irfan Guzel

Comparison of DNA isolation methods from mammalian sperm cells and development of a new protocol

Sperm DNA tightly packed with protamines makes the DNA isolation procedure from sperm cells long and laborious. Cell lysis is also a challenging step because of the disulfide bonds-rich membranes of the sperm cells. In this study we aimed to evaluate potential rapid DNA isolation protocols to isolate DNA from mammalian sperm cells, and develop an easy, rapid, and cost-effective protocol for sperm DNA isolation which can be used in molecular biology and diagnostics. Sperm samples were collected from seven adult rats. Our developed protocol included Proteinase K and small amount of β-mercaptoethanol (βME) for cell lysis. A modified salting-out technique was then employed to collect DNA. Alternative protocols involving InstaGene matrix and cell sonication techniques were also applied to achieve DNA isolation. Concentration of the DNA yield was measured, and the degradation of DNA was checked using agarose gel electrophoresis. The intactness of the DNA yield was assessed and validated using polymerase chain reaction (PCR) and capillary gel electrophoresis techniques. The lysis of the cells and high-quality DNA yield have only been achieved using our developed optimized protocol. To confirm the quality of DNA for assays, PCR product was synthesized for rat actin β (RActβ) gene and then analyzed using capillary gel electrophoresis. A strong peak at right m/z value for the amplicon was obtained. We described an improved protocol over the previous methods suggesting the use of combined commercial kits and long incubation times. Degradation-free DNA was produced in a relatively rapid protocol (90 minutes) using the equipment and supplies common to most research and clinical laboratories.

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Balhorn R. The protamine family of sperm nuclear proteins. Genome Biology. 2007;8:227.
Oliva R, Dixon GH. Vertebrate protamine genes and the histone-toprotamine replacement reaction. Progress Nucleic Acid Res Molecular Biol. 1991;40:25-94.
Castillo J, Amaral A, Oliva R. Sperm nuclear proteome and its epigenetic potential. Andrology. 2014;2:326-38.
de Boer P, de Vries M, Ramos L. A mutation study of sperm head shape and motility in the mouse: lessons for the clinic. Andrology. 2015;3:174-202.
Houshdaran S, Cortessis VK, Siegmund K, et al. Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm. PLoS One. 2007;2:e1289.
Takasaki N, Tachibana K, Ogasawara S, et al. A heterozygous mutation of GALNTL5 affects male infertility with impairment of sperm motility. Proc Natl Acad Sci U S A. 2014;111:1120-5.
Goldenberger D, Perschil I, Ritzler M, et al. A simple "universal" DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification. PCR Methods Appl. 1995;4:368-70.
Santella RM. Approaches to DNA/RNA Extraction and whole genome amplification. Cancer Epidemiol Biomarkers Prev. 2006;15:1585-7.
Walsh PS, Metzger DA, Higuchi R. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques. 1991;10:506-13.
Kim SH, Jeong HS, Kim YH, et al. Evaluation of DNA extraction methods and their clinical application for direct detection of causative bacteria in continuous ambulatory peritoneal dialysis culture fluids from patients with peritonitis by using broad-range PCR. Ann Lab Med. 2012;32:119-25.
Hoff-Olsen P, Mevag B, Staalstrom E, et al. Extraction of DNA from decomposed human tissue. An evaluation of five extraction methods for short tandem repeat typing. Forensic Sci Int. 1999;105:171-83.
Hossain AM, Rizk B, Behzadian A, Thorneycroft IH. Modified guanidinium thiocyanate method for human sperm DNA isolation. Mol Hum Reprod. 1997;3:953-6.
Griffin J. Methods of sperm DNA extraction for genetic and epigenetic studies. Methods Mol Biol. 2013;927:379-84.
Wu H, de Gannes MK, Luchetti G, et al. Rapid method for the isolation of mammalian sperm DNA. Biotechniques. 2014;58:293-300.
Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Research. 1988;16:1215.
Sambrook J, Russell DW. Fragmentation of DNA by sonication. CSH Protoc. 2006;2006.
Knierim E, Lucke B, Schwarz JM, et al. Systematic comparison of three methods for fragmentation of long-range PCR products for next generation sequencing. PLoS One. 2011;6:e28240