使用逆嵌套PCR检测含有整合的乙型肝炎病毒DNA的肝细胞克隆

StephenW

Methods Mol Biol. 2017;1540:97-118.
Detection of Hepatocyte Clones Containing Integrated Hepatitis B Virus DNA Using Inverse Nested PCR.Tu T1,2,3, Jilbert AR4.
Author information

• 1Liver Cell Biology Laboratory, Centenary Institute, Sydney, NSW, 2050, Australia.
• 2Sydney Medical School, University of Sydney, Sydney, NSW, 2050, Australia.
• 3Department of Molecular and Cellular Biology, School of Biological Sciences, University of Adelaide, Adelaide, SA, 5005, Australia.
• 4Department of Molecular and Cellular Biology, School of Biological Sciences, University of Adelaide, Adelaide, SA, 5005, Australia. [email protected].
  

AbstractChronic hepatitis B virus (HBV) infection is a major cause of liver cirrhosis and hepatocellular carcinoma (HCC), leading to ~600,000 deaths per year worldwide. Many of the steps that occur during progression from the normal liver to cirrhosis and/or HCC are unknown. Integration of HBV DNA into random sites in the host cell genome occurs as a by-product of the HBV replication cycle and forms a unique junction between virus and cellular DNA. Analyses of integrated HBV DNA have revealed that HCCs are clonal and imply that they develop from the transformation of hepatocytes, the only liver cell known to be infected by HBV. Integrated HBV DNA has also been shown, at least in some tumors, to cause insertional mutagenesis in cancer driver genes, which may facilitate the development of HCC. Studies of HBV DNA integration in the histologically normal liver have provided additional insight into HBV-associated liver disease, suggesting that hepatocytes with a survival or growth advantage undergo high levels of clonal expansion even in the absence of oncogenic transformation. Here we describe inverse nested PCR (invPCR), a highly sensitive method that allows detection, sequencing, and enumeration of virus-cell DNA junctions formed by the integration of HBV DNA. The invPCR protocol is composed of two major steps: inversion of the virus-cell DNA junction and single-molecule nested PCR. The invPCR method is highly specific and inexpensive and can be tailored to DNA extracted from large or small amounts of liver. This procedure also allows detection of genome-wide random integration of any known DNA sequence and is therefore a useful technique for molecular biology, virology, and genetic research.


KEYWORDS: Clonal expansion; Insertional mutagenesis; Integrated DNA; Inverse nested PCR; Virus–cell DNA junction

PMID:27975311DOI:10.1007/978-1-4939-6700-1_9

StephenW

方法。 2017; 1540：97-118。
使用逆嵌套PCR检测含有整合的乙型肝炎病毒DNA的肝细胞克隆。
Tu T1,2,3，Jilbert AR4。
作者信息

    1Liver Cell Biology Laboratory，Centenary Institute，Sydney，NSW，2050，Australia。
    悉尼大学悉尼医学院，NSW，2050，澳大利亚。
    3分子和细胞生物学，阿德莱德大学生物科学学院，阿德莱德，SA，5005，澳大利亚。
    4分子和细胞生物学，生物科学学院，阿德莱德大学，阿德莱德，SA，5005，澳大利亚。 [email protected]。

抽象

慢性乙型肝炎病毒（HBV）感染是肝硬化和肝细胞癌（HCC）的主要原因，导致全世界每年约600,000例死亡。在从正常肝进展到肝硬化和/或HCC期间发生的许多步骤是未知的。将HBV DNA整合入宿主细胞基因组中的随机位点作为HBV复制循环的副产物，并在病毒和细胞DNA之间形成独特的连接。综合HBV DNA的分析表明，HCC是克隆的，意味着它们从肝细胞的转化发展而来，肝细胞是已知被HBV感染的唯一肝细胞。还已经显示整合的HBV DNA，至少在一些肿瘤中，在癌症驱动基因中引起插入诱变，这可以促进HCC的发展。在组织学正常肝脏中HBV DNA整合的研究提供了对HBV相关的肝脏疾病的另外的了解，表明具有存活或生长优势的肝细胞即使在没有致癌性转化的情况下也经历高水平的克隆扩增。在这里我们描述反嵌套PCR（invPCR），一种高度敏感的方法，允许检测，测序和枚举由HBV DNA整合形成的病毒细胞DNA连接。 invPCR方案由两个主要步骤组成：病毒 - 细胞DNA连接反转和单分子嵌套PCR。 invPCR方法是高度特异性和便宜的，并且可以适合从大量或少量肝提取的DNA。该程序还允许检测任何已知DNA序列的全基因组随机整合，因此是分子生物学，病毒学和遗传研究的有用技术。
关键词：

克隆扩增;插入诱变;集成DNA;反嵌套PCR;病毒 - 细胞DNA连接

PMID：
    27975311
DOI：
    10.1007 / 978-1-4939-6700-1_9

neilhbver

可以检测有多少整合到肝细胞的hbv？希望能结合年龄段研究一下。