- Title
- Improved methods of DNA extraction from human spermatozoa that mitigate experimentally-induced oxidative DNA damage
- Creator
- Xavier, Miguel J.; Nixon, Brett; Roman, Shaun D.; Aitken, Robert John
- Relation
- PLOS One Vol. 13, Issue 3
- Publisher Link
- http://dx.doi.org/10.1371/journal.pone.0195003
- Publisher
- Public Library of Science
- Resource Type
- journal article
- Date
- 2018
- Description
- Current approaches for DNA extraction and fragmentation from mammalian spermatozoa provide several challenges for the investigation of the oxidative stress burden carried in the genome of male gametes. Indeed, the potential introduction of oxidative DNA damage induced by reactive oxygen species, reducing agents (dithiothreitol or beta-mercaptoethanol), and DNA shearing techniques used in the preparation of samples for chromatin immunoprecipitation and next-generation sequencing serve to cofound the reliability and accuracy of the results obtained. Here we report optimised methodology that minimises, or completely eliminates, exposure to DNA damaging compounds during extraction and fragmentation procedures. Specifically, we show that Micrococcal nuclease (MNase) digestion prior to cellular lysis generates a greater DNA yield with minimal collateral oxidation while randomly fragmenting the entire paternal genome. This modified methodology represents a significant improvement over traditional fragmentation achieved via sonication in the preparation of genomic DNA from human spermatozoa for downstream applications, such as next-generation sequencing. We also present a redesigned bioinformatic pipeline framework adjusted to correctly analyse this form of data and detect statistically relevant targets of oxidation.
- Subject
- DNA damage; sperm; oxidative damage; reduced fertility
- Identifier
- http://hdl.handle.net/1959.13/1401694
- Identifier
- uon:34948
- Identifier
- ISSN:1932-6203
- Rights
- Copyright: © 2018 Xavier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
- Language
- eng
- Full Text
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