前病毒
套式聚合酶链反应
长终端重复
生物
DNA
底漆二聚体
聚合酶链反应
分子生物学
实时聚合酶链反应
铝元素
病毒学
反聚合酶链反应
遗传学
基因组
基因
人类基因组
多重聚合酶链反应
作者
Audrey Brussel,Olivier Delelis,Pierre Sonigo
出处
期刊:Humana Press eBooks
[Humana Press]
日期:2005-06-08
卷期号:: 139-154
被引量:35
标识
DOI:10.1385/1-59259-907-9:139
摘要
An improved Alu-long terminal repeat (LTR) polymerase chain reaction (PCR) assay is described for the quantification of integrated HIV-1 DNA in infected cells. The method includes generation of an infected cell line containing numerous randomly distributed HIV-1 integrated DNA for the construction of the DNA standard and a two-step real-time PCR assay in which the first-round PCR amplifies the DNA sequence between the HIV-1 LTR and the nearest chromosomal Alu element, and the nested PCR specifically amplifies PCR products from the first-round PCR. This assay allows us to quantify proviral DNA with both accuracy and high sensitivity (six proviruses within 50,000 cell equivalents) and exhibits a broad range of quantification spanning 5 log10 provirus copies. This Alu-LTR-based real-time nested PCR assay may be particularly useful to quantify integrated HIV-1 DNA in patients. It may also allow for the precise study of integration of HIV-1 DNA or HIV-1 based lentiviral vectors and may be a valuable tool to test future inhibitors of integration.
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