基于新型磁分离技术的高分辨率DNA片段筛选技术

doi:10.13560/j.cnki.biotech.bull.1985.2020-1478

摘要/Abstract

摘要：

游离DNA作为液体活检的目标检测物,能够用于疾病的检测,但游离DNA在样本中的含量低,且片段差异小,常规方法很难实现对特定片段长度游离DNA的筛选。这种基于新型磁分离技术对游离DNA进行高分辨率筛选的方法,使用自主合成的两种不同修饰的磁珠,在沉降剂的作用下,能够实现长短片段DNA的分别回收。通过对血浆样本中游离DNA进行富集,实现了150 bp以下DNA片段的富集,并经由临床样本验证,这一技术能够实现母体血浆样本中胎儿游离DNA的富集,可在无创产前诊断样本处理环节提前检测窗口、提高检测灵敏度等。DNA片段筛选在基因测序领域是不可或缺的环节,高分辨率筛选可降低下游测序深度、节约测序成本等,可应用于肿瘤的早期筛查、无创产前诊断和免疫缺陷型疾病筛查领域。

关键词: DNA片段筛选, 新型磁分离技术, 游离DNA, 液体活检, 无创产前诊断

Abstract:

Cell-free DNA（cfDNA）as one of the target detection substance for liquid biopsy can be used for disease detection. However,the concentration of cfDNA in a sample is low and difficult in fragment discrimination,thus it is difficult to separate cfDNA within specific fragment length by conventional methods. In this method for high-resolution cfDNA screening based on new magnetic separation technology,two different modified magnetic beads synthesized by ourself were used to separately have recovery of long- and short-fragments DNA under the action of sedimentation agent. This technology achieved the enrichment of cfDNA below 150 bp in plasma samples. We verified by clinical samples that this technology achieved the enrichment of fetal free DNA in maternal plasma. It could be applied in non-invasive prenatal testing（NIPT）to bring forward early detection and improve detection sensitivity,etc. DNA fragment screening is an indispensable part in the field of gene sequencing,and high resolution screening can reduce downstream sequencing depth and save sequencing cost,and applied in tumors screening,non-invasive prenatal diagnosis and immunodeficiency diseases screening.

Key words: DNA fragment screening, novel magnetic separation technique, cfDNA, liquid biopsy, non-invasive prenatal testing

刘莹, 程立, 张博. 基于新型磁分离技术的高分辨率DNA片段筛选技术[J]. 生物技术通报, 2021, 37(10): 257-265.

LIU Ying, CHENG Li, ZHANG Bo. High-resolution DNA Screening Based on Novel Magnetic Separation Technology[J]. Biotechnology Bulletin, 2021, 37(10): 257-265.

图/表 6

图1 富集短片段DNA的流程图

Fig. 1 Workflow of short DNA fragments enrichment

表1 引物和探针信息表

Table 1 Information of primers and probes

引物名称Primer	引物序列Sequence
Y染色体正向引物（qPCR）	CATTCTCAAGCAAAACATGG
Y染色体反向引物（qPCR）	CAGCAGTAGAGCAGTCAG
Y染色体探针（qPCR）	CGTTGACTACTTGCCCT
Y染色体正向引物（ddPCR）	CATTCTCAAGCAAAACATGG
Y染色体反向引物（ddPCR）	CAGCAGTAGAGCAGTCAG
Y染色体探针（ddPCR）	CGTTGACTACTTGCCCT
X染色体正向引物（ddPCR）	AGTCACACCCACTTGTT
X染色体反向引物（ddPCR）	GGGCCATACAATCTGTTG
X染色体探针（ddPCR）	CTCTGACCTGGTAGCAC

图2 cfDNA短片段富集的模型体系验证 A :富集前后DNA片段分布;B :富集前后150 bp以下DNA片段占比变化;C :富集前后cfDNA主峰平均长度变化

Fig.2 Verification of short DNA fragments enrichment A: DNA fragments distribution before and after enrichment. B: DNA fragments distribution（below 150 bp）before and after enrichment. C: Average length of cfDNA main peak before and after entichment

图3 ddPCR验证结果 A :富集前后ddPCR对X、Y染色体拷贝数检测流程示意图;B :富集前后ddPCR荧光信号示意图;C :21个男性胎儿孕妇血浆样本富集前后Y染色体比例变化;D :21个男性胎儿孕妇血浆样本富集前后Y染色体拷贝数变化倍数

Fig.3 Enrichment efficiency evaluation by ddPCR A: Workflow of copy number calculation for ChrX and ChrY by ddPCR before and after enrichment. B: Fluorescence signal of sample before and after enrichment. C: ChrY% statistics of 21 samples from pregnant women with male fetus. D: ChrY% fold change for 21 samples from pregnant women with male fetus

图4 优化后富集短片段DNA的流程图

Fig.4 Optimized workflow of short DNA fragments enrichment

图5 优化后胎牛血清cfDNA短片段的富集 A :二次筛选后富集前后DNA片段分布变化;B :二次筛选两步长片段分离DNA片段分布,两步长片段分离起峰位置均在150 bp左右;C :二次筛选富集前后150 bp以下DNA片段占比变化;D :二次筛选富集前后cfDNA主峰平均长度变化

Fig. 5 Enrichment of short cfDNA fragments with fetal bovine serum by optimized workflow A: Changes in DNA fragments distribution before and after 2-step selection enrichment. B: Fragment distribution by 2-time selection and 2-step long fragments DNA separation with the peak beginning position at 150 bp. C: Proportion of DNA below 150 bp before and after enrichment. D: Average length change of cfDNA main peak

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