PCR-based procedures to isolate insertion sites of DNA elements
PCR-based procedures to isolate insertion sites of DNA elements
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DOI：
10.2144/000112474
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发表时间：
2007-07-01
期刊：
影响因子：
2.7
通讯作者：
Largaespada, David A.
中科院分区：
文献类型：
--
作者：
Yin, Bin;Largaespada, David A.
在过去的几年中，逆转录病毒插入突变已卓有成效地应用于寻找与肿瘤发生有关的基因/途径。用于鉴定前病毒插入位点的技术对于完成这些项目至关重要。尽管已经描述了各种方法，但是需要对现有方法进行改进以恢复用于癌症基因发现的每个可能的插入位点，即所谓的饱和分析。在这里，我们已经描述了两个连接介导的PCR变体，SplinkTA-PCR（STA-PCR）和SplinkBlunt-PCR的发展，在逆转录病毒诱导的白血病插入位点的有效隔离。我们的研究结果表明，这两个协议是互补的，彼此，他们更好地结合使用，以获得最大的克隆效率。这些协议是易于使用，可靠和有效的，并很容易适用于大规模克隆插入位点的前病毒和其他整合的DNA元件，以及用于检测和克隆的差异插入独特的耐药细胞。
During the past several years, retroviral insertional mutagenesis has been fruitfully applied to search for genes/pathways involved in tumorigenesis. Techniques used to identify proviral insertion sites are critical for fulfilling these projects. Although a variety of approaches have been described, an improvement over existing methods is required to recover every possible insertion site for cancer gene discovery, so-called saturation analysis. Here, we have described the development of two ligation-mediated PCR variants, SplinkTA-PCR (STA-PCR) and SplinkBlunt-PCR, for efficient isolation of insertion sites in retrovirus-induced leukemia. Our results demonstrated that these two protocols are complementary to each other and that they are better employed in combination for maximal cloning efficiency. These protocols are easy-to-use, reliable and efficient, and are readily applicable to large-scale cloning of insertion sites of provirus and other integrated DNA elements, as well as for detection and cloning of differential insertions unique to drug-resistant cells.