Tozaki Teruaki, Ohnuma Aoi, Kikuchi Mio, Ishige Taichiro, Kakoi Hironaga, Hirota Kei-Ichi, Hamilton Natasha A, Kusano Kanichi, Nagata Shun-Ichi
Genetic Analysis Department, Laboratory of Racing Chemistry, Tochigi 320-0851, Japan.
Equine Genetics Research Centre, Racing Australia, NSW 2337, Australia.
J Equine Sci. 2020;31(4):75-83. doi: 10.1294/jes.31.75. Epub 2020 Dec 18.
Gene doping is prohibited in horseracing and equestrian sports. In previous studies, we developed non-targeted transgene and genome editing detection methods based on whole genome resequencing (WGR) using genomic DNA extracted from whole blood. In this study, we aimed to develop a WGR method using DNA extracts from hair roots. Hair roots are a preferred substrate because their collection is less invasive than blood collection. Hair is also easier to store for long periods of time. Although almost all genomic DNA extracted from hair root samples stored for years at room temperature was degraded, the quality of genomic DNA from samples stored for years at refrigerated temperatures (4-8°C) was maintained. High-molecular-weight genomic DNA was isolated from hair roots using a magnetic silica beads method of extraction, enabling WGR from horsehair root extracts. Nucleotide sequencing results and numbers of single-nucleotide polymorphisms and insertions/deletions concurred with those previously reported for WGR of DNA extracted from whole blood. Therefore, we consider that storing hair samples at refrigerated temperatures prevents degradation of DNA, allowing the detection of gene doping in these samples based on WGR. It is likely this finding will also have a deterrent effect, as it is now possible to test horses with archived samples even if they or their parents are deceased. To our knowledge, this is the first report employing WGR on horsehair roots stored for a long term.
基因兴奋剂在赛马和马术运动中是被禁止的。在之前的研究中，我们基于全基因组重测序（WGR）开发了非靶向转基因和基因组编辑检测方法，使用从全血中提取的基因组DNA。在本研究中，我们旨在开发一种使用发根DNA提取物的WGR方法。发根是一种优选的样本来源，因为采集发根比采集血液的侵入性更小。毛发也更容易长期保存。尽管从室温下储存数年的发根样本中提取的几乎所有基因组DNA都已降解，但在冷藏温度（4-8°C）下储存数年的样本中基因组DNA的质量得以保持。使用磁珠法从发根中分离出高分子量基因组DNA，从而能够对马发根提取物进行WGR。核苷酸测序结果以及单核苷酸多态性和插入/缺失的数量与先前报道的从全血中提取的DNA进行WGR的结果一致。因此，我们认为在冷藏温度下储存毛发样本可防止DNA降解，从而能够基于WGR检测这些样本中的基因兴奋剂。这一发现可能也会产生威慑作用，因为即使马匹或其父母已经死亡，现在也可以用存档样本对马匹进行检测。据我们所知，这是第一份对长期储存的马发根进行WGR的报告。
