Construction and use of spotted large-insert clone DNA microarrays for the detection of genomic copy number changes.
Construction and use of spotted large-insert clone DNA microarrays for the detection of genomic copy number changes.
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DOI：
10.1038/nprot.2007.53
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发表时间：
2007
期刊：
影响因子：
14.8
通讯作者：
Carter, Nigel P.
中科院分区：
文献类型：
--
作者：
Fiegler, Heike;Redon, Richard;Carter, Nigel P.
基于微阵列的比较基因组杂交已经成为分析整个人类基因组DNA拷贝数变化的广泛方法。使用大插入克隆构建微阵列的最初方法需要从大规模培养物中制备DNA。当扩展到更高分辨率阵列所需的克隆数量时，这很快就变成了一个昂贵且耗时的过程。为了克服这个问题，已经开发了几种基于聚合酶链式反应的策略，以使从少量克隆DNA构建阵列成为可能。在这里，我们描述了使用一种特定的简并寡核苷酸聚合酶链式反应策略构建由人类特异性大插入克隆(40-200kb)组成的微阵列。此外，我们还描述了使用手动和自动程序和方法进行阵列分析的阵列杂交。根据克隆库的可用性，本文中描述的技术和协议可以很容易地适用于其他物种。根据我们的方案，从标记DNA到扫描杂交玻片大约需要3天的时间。
Microarray-based comparative genomic hybridization has become a widespread method for the analysis of DNA copy number changes across the human genome. Initial methods for microarray construction using large-insert clones required the preparation of DNA from large-scale cultures. This rapidly became an expensive and time-consuming process when expanded to the number of clones needed for higher resolution arrays. To overcome this problem, several PCR-based strategies have been developed to enable array construction from small amounts of cloned DNA. Here, we describe the construction of microarrays composed of human-specific large-insert clones (40–200 kb) using a specific degenerate oligonucleotide PCR strategy. In addition, we also describe array hybridization using manual and automated procedures and methods for array analysis. The technology and protocols described in this article can easily be adapted for other species dependent on the availability of clone libraries. According to our protocols, the procedure will take approximately 3 days from labeling the DNA to scanning the hybridized slides.
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