新生儿疾病筛查血片微量DNA提取方法的建立及其在甲基化检测中的应用

Isolation of human genomic DNA from archival dried blood spots for neonatal disease screening and its application to methylation detection

目的 建立新生儿疾病筛查血片DNA提取的方法,探索其在甲基化检测中的应用.方法 依据性别随机将新生儿出生时的血片样本20例分为2组：传统法组（10例）、改良试剂盒法组（10例）.从DNA浓度、完整性和是否适应于聚合酶链反应（PCR）研究,对抽提的DNA质量进行评估.进一步对DNA做重亚硫酸盐处理,采用甲基化特异性PCR（MSP）法检测瘦素（Leptin）、肿瘤坏死因子-α（TNF-α）基因启动子区域甲基化水平变化.结果 改良试剂盒法抽提的DNA浓度为（5.70 ±0.81） mg/L,显著优于传统法[（3.50±0.45）mg/L]（=2.79,P＜0.05）;生化分析仪分析显示DNA片段完整性更好.琼脂糖凝胶电泳显示可以扩增出有效的18S基因PCR片段,说明抽提的DNA可以用于PCR相关实验研究;MSP结果显示不同血片样本甲基化Leptin、TNF-α基因启动子区域甲基化模式不同.结论 改良试剂盒方法可以简便有效地从干血片中提取DNA,而且抽提的DNA可以较好地用于基因甲基化研究;为从DNA甲基化的角度揭示疾病的发生,提供了新的研究方案和技术方法.

Objective To establish an effective DNA isolation method for neonatal disease screening,so as to explore its application to the methylation detection.Methods The 20 dried blood spots samples were randomly divided into 2 groups according to the gender：the traditional method group （n =10） and the improved kit method group（n =10）.The DNA quality was evaluated based on its concentration,integrity and whether it could be used in polymerase chain reaction （PCR）.These DNA samples with or without bisulfite treatment were used as template in the methylation-specific polymerase chain reaction （MSP）.The methylation levels of Leptin and tumor necrosis factor-α （TNF-α） gene promoter region were detected.Results DNA concentration of the improved kit method [（5.70 ± 0.81） mg/L] was significantly higher than that of the traditional method [（3.50 ± 0.45） mg/L] （t =2.79,P 〈 0.05）,and biochemical analyzer analysis showed a better DNA integrity.Agarose gel electrophoresis revealed that 18S gene fragment could be successfully amplified by PCR method,suggesting its potential application to PCR study.MSP results showed different DNA methylation levels of Leptin and TNF-α genes promoter regions from various samples.Conclusions The improved kit method can effectively extract DNA from dried blood spots samples,and these DNA can be used in methylation research.The study can provide a new research direction and technical method to reveal the pathogenesis of disease from the perspective of DNA methylation.