HCV阴性者外周血单核细胞的染色体外环状DNA中与HCV同源的DNA序列（英文）

论文摘要

目的:研究与丙型肝炎病毒（HCV）基因RNA序列同源的人染色体外环状DNA（eccDNA）序列。创新点:首次从HCV阴性者eccDNA中检测到HCV5’-非编码区（5’-NCR）基因组RNA序列,验证了我们的假设:HCV同源DNA序列存在于人的外周血单核细胞的eccDNA组分中。方法:用分离的eccDNA进行HCV特异的聚合酶链反应（PCR）,采用核苷酸序列同源性搜索分析软件（BLASTn）对测序结果进行比对分析,并检测其甲基化模式。结论:实验结果证实了我们的假设:即部分HCV 5’-NCR基因组RNA序列存在于外周血单核细胞的eccDNA组分。同时,甲基化分析结果显示了个体间的甲基化模式所代表的受遗传调控的表观遗传特征。

论文目录

1 Introduction

2 Results

2.1 Computational approaches by alignmentsearch with BLASTn and the 5'-NCR of HCV in human genomic DNA

2.2 Experimental approaches

2.2.1 Isolation of eccDNA from whole cellular DNA

2.2.2 PCR tests

2.2.3 Testing for methylation of the sequence section selected by the primers used

2.2.4 Test for human endogenous retrovirus type K （HERV-K） sequences in the eccDNA fraction

3 Discussion

3.1 HCV genomic sequences contained in the human genome

3.2 Extrachromosomal DNA:ecc DNA and its molecular features

3.3 Facts and suppositions about the genesis of ecc DNA

3.4 Patterns of methylation and epigenetics

4 Conclusions

Contributors

Compliance with ethics guidelines

List of electronic supplementary materials

Materials and methods, supporting online information for protocols

1.Subjects and Isolation of peripheral blood mononuclear cells （PBMCs） from human plasma

2.Isolation of the whole DNA from PBMCs

3.Preparation of eccDNA from the whole DNA extracted from PBMCs

4. Isolation of gel purified PSAD-resistant DNA/ecc DNA:

5. Protocol to check for patterns of methylation in the HCV-DNA sequences of the HCV 5'-NCR targeted here:the pre-PCR digestion protocol with restriction endonucleases prior to the PCR with gene-specific primers

6. PCR assays to test for the PSAD-resistant DNA/eccDNA with HCV gene-specific primers

7. Details of the PCR protocols

8. Cloning of the amplicons after PCR for sequencings

9. Sequencing

1 0. Alignments by BLASTn

1 1. Protocol used to demonstrate the relative amount of the PSAD-resistant eccDNA fraction

References

文章来源

类型: 期刊论文

作者: Reinhard H.DENNIN,Jian-er WO

关键词: 丙型肝炎病毒,非编码区,人类基因组,染色体外,环状,甲基化模式

来源: Journal of Zhejiang University-Science B（Biomedicine & Biotechnology） 2019年08期

年度: 2019

分类: 工程科技Ⅱ辑,基础科学,医药卫生科技

专业: 生物学,基础医学

单位: Formerly Department of Infectious Diseases and Microbiology, University of Lübeck,University Hospital Schleswig-Holstein,State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Institute of Infectious Diseases,the First Affiliated Hospital, School of Medicine, Zhejiang University

基金: Project supported by the Exchange Scholarship Programs of the Landesregierung Schleswig-Holstein,Germany,for Jian-er WO

分类号: R373.21

页码: 637-646+1-4

总页数: 14

文件大小: 877K

下载量: 52

HCV阴性者外周血单核细胞的染色体外环状DNA中与HCV同源的DNA序列（英文）

论文摘要

论文目录

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