Quick recovery and characterization of cell-free DNA in seminal plasma of normozoospermia and azoospermia: implications for non-invasive genetic utilities
Quick recovery and characterization of cell-free DNA in seminal plasma of normozoospermia and azoospermia: implications for non-invasive genetic utilities
复制标题
正常精子症和无精子症精浆中游离 DNA 的快速恢复和表征：对非侵入性遗传实用程序的影响。
DOI：
10.1038/aja.2009.65
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发表时间：
2009-11-01
影响因子：
2.9
通讯作者：
Xiong, Cheng-Liang
中科院分区：
文献类型：
--
作者：
Li, Hong-Gang;Huang, Shi-Yun;Xiong, Cheng-Liang
建立了一种快速、可靠的精液游离DNA（cfsDNA）回收方法。cfsDNA量的一式三份值之间的低变化（低于15%）验证了我们的cfsDNA回收方法的再现性。通过过滤和离心获得的精浆之间相似的cfsDNA产率和大小分布证实了cfsDNA的存在。为了研究cfsDNA的一般特征，分别通过实时PCR和电泳分析来自正常精子和无精子精液的cfsDNA的定量和大小分布。正常精子症（n= 11）精液中的cfsDNA浓度为1.34±0.65 μg mL− 1，而无精子症（2.56±1.43 μg mL− 1，n= 9）中观察到较高的cfsDNA浓度。在正常精子和无精子的样本中，观察到DNA片段的连续分布，范围从0.1 kb到15 kb，并且有多个180 bp的片段。在一些无精子症患者的样本中出现了明显的DNA梯状断裂，提示cfsDNA部分来源于凋亡细胞。对36例已知Y染色体微缺失信息的无精子症患者的cfsDNA进行多重PCR和sY114（1450bp）的PCR扩增。cfsDNA的所有多重PCR反应均成功扩增，并提供与白细胞DNA相同的结果。PCR扩增sY114，得到1450bp的扩增产物。我们的数据表明cfsDNA在寻找生物标志物或诊断程序中的潜在用途。
We established a quick and reliable method for recovering cell-free seminal DNA (cfsDNA), by using the binding-washing-elution procedure on the DNA purification column. Low variations (below 15%) among the triplicate values of cfsDNA quantity verified the reproducibility of our cfsDNA recovery method. Similar cfsDNA yield and size distribution between seminal plasma acquired by filtration and centrifugation confirmed the presence of cfsDNA. To investigate the general characterization of cfsDNA, the quantitation and size distribution of cfsDNA from normozoospermic and azoospermic semen were analyzed by real-time PCR and electrophoresis, respectively. CfsDNA concentration in semen with normozoospermia (n= 11) was 1.34±0.65 μg mL− 1, whereas a higher cfsDNA concentration was observed in azoospermia (2.56±1.43 μg mL− 1, n= 9). The continuous distribution of DNA fragments ranging from∼ 1 kb to 15 kb and a spectrum of multiples of 180-bp fragments were observed in each normozoospermic and azoospermic sample. Distinct characteristic DNA ladder fragmentations in some azoospermic samples implicated that cfsDNA originate partly from apoptotic cells. CfsDNAs of 36 selected azoospermic patients with known information of Y chromosome microdeletion were subjected to the same microdeletion analysis by multiplex PCR and PCR amplification of sY114 (1 450 bp). All multiplex PCR reactions with cfsDNA amplified successfully and provided the same result as leukocyte DNA. PCR amplification of sY114 gave a 1 450-bp amplicon as expected. Our data suggested the potential use of cfsDNA in search of biomarker or diagnostic procedures.