摘要
目的超短片段游离DNA(ultra-short cell free DNA,uscfDNA)是一种新发现的cfDNA,具备疾病筛查分子标志物潜能。本项目拟通过优化孕妇外周血cfDNA提取方法,实现从孕妇外周血中高效分离和回收uscfDNA。方法回顾性收集2021年11月至2024年8月于深圳市龙岗区妇幼保健院接受无创产前筛查(noninvasive prenatal screening,NIPS)孕周为12~37周检测的135例孕妇自愿者检测后剩余标本和临床资料。通过使用高比例磁珠和高比例醇类对常规提取方法和回收方式进行优化以提升cfDNA提取效果,将磁珠法与有机试剂抽提和醇沉法相结合来提高纯化效果。结果通过对常规提取方法的优化,从孕妇外周血中获取到50bp左右的单链DNA的uscfDNA,在总cfDNA中占21%~96%。通过评估uscfDNA在基因组中的分布情况,发现其主要在启动子等调节区域富集。结论本研究通过优化cfDNA提取纯化方法,实现了uscfDNA的高效提取。该方法或可为基于围产领域uscfDNA研究提供帮助。
Objective Ultra-short cell-free DNA(uscfDNA)is a recently identified form of cfDNA with potential as a molecular biomarker for disease screening.The project aimed to optimise the methods for extracting cfDNA from maternal peripheral blood,to effectively isolate and recover uscfDNA.Methods Residual plasma samples and associated clinical data were collected from 135pregnant women undergoing non-invasive prenatal screening(NIPS)between 12 and 37 weeks of gestation at Shenzhen Longgang Maternal and Child Health Hospital between November 2021 and August 2024.We optimised conventional extraction and recovery protocols using high-concentration magnetic beads and alcohols to enhance cfDNA extraction efficiency.The combination of magnetic bead-based extraction,organic solvent extraction,and ethanol precipitation improved purification efficiency.Results Using these optimised methods,we successfully extracted uscfDNA from maternal peripheral blood.The uscfDNA was characterised as predominantly single-stranded DNA fragments of approximately 50 base pairs,accounting for 21-96% of the total cfDNA,with variation observed among samples.Analysis of uscfDNA distribution across the genome revealed significant enrichment in regulatory regions such as promoters.Conclusion This study successfully extracted uscfDNA by optimising the extraction and purification of cfDNA,providing an improved approach that could facilitate further research into uscfDNA in the perinatal field.
作者
谢伟洁
曹宪振
钟佳通
温丽娟
胡亮
刘维强
Xie Weijie;Cao Xianzhen;Zhong Jiatong;Wen Lijuan;Hu Liang;Liu Weiqiang(Central Laboratory,Affiliated Shenzhen Women and Childrens Hospital(Longgang)of Shantou University Medical College(Longgang Maternity and Child healthcare Hospital o f Shenzhen City),Shenzhen,Guangdong 518172,China)
出处
《中国产前诊断杂志(电子版)》
2025年第4期47-51,共5页
Chinese Journal of Prenatal Diagnosis(Electronic Version)
基金
深圳市自然科学基金项目(JCYJ20230807141908017)
深圳市龙岗区科创局项目(LGKCYLWS2022013)。
关键词
超短片段游离DNA
提取方法
磁珠
单链DNA
Ultra-short cell-free DNA
Extraction method
Magnetic beads
Single-stranded DNA(ss DNA)
