Improved methods of DNA extraction from human spermatozoa that mitigate experimentally-induced oxidative DNA damage

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, red...

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Bibliographic Details
Published in:PloS one 2018-03, Vol.13 (3), p.e0195003-e0195003
Main Authors: Xavier, Miguel J, Nixon, Brett, Roman, Shaun D, Aitken, Robert John
Format: Article
Language:English
Subjects:
Biology
Biology and Life Sciences
Chromatin
Damage
Deoxyribonucleic acid
Dithiothreitol
DNA
DNA damage
DNA fragmentation
DNA sequencing
Fragmentation
Gametes
Gene sequencing
Genetic aspects
Genomes
Immunoprecipitation
Lysis
Methods
Nuclease
Nucleic acid isolation
Oxidation
Oxidative stress
Oxygen
Physiological aspects
Reactive oxygen species
Reducing agents
Research and analysis methods
Shearing
Sonication
Sperm
Spermatozoa
Statistical analysis
Statistical methods
Target detection
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0195003