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使用微阵列检测致病细菌基因组DNA的全基因组扩增方法比较

Comparison of whole genome amplification methods for detecting pathogenic bacterial genomic DNA using microarray.

作者信息

Uda Akihiko, Tanabayashi Kiyoshi, Fujita Osamu, Hotta Akitoyo, Yamamoto Yoshie, Yamada Akio

机构信息

Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo 162-8640, Japan.

出版信息

Jpn J Infect Dis. 2007 Nov;60(6):355-61.

Abstract

The genetic diagnosis of pathogenic agents using microarrays has the advantage of high-throughput detection, but a relatively large amount of DNA sample is required. To obtain a sufficient amount of DNA for molecular diagnoses, several whole genome amplification (WGA) methods have been proposed. In this study, using Francisella tularensis and Escherichia coli as models, we compared four WGA methods in terms of their efficiency of amplification of whole genomic DNA in order to identify the most suitable method for preparing DNA to be used for microarray analysis. It was possible to obtain more than 1.5 microg of products from 10 ng of F. tularensis and E. coli genomic DNA using four methods, but biases in the amplification of bacterial genes were least prominent in the multiple displacement amplification (MDA) or OmniPlex WGA. When the amplified DNAs were applied to microarray slides consisting of 32 different genes probes, DNAs amplified by Phi29 v2 of MDA and OmniPlex WGA showed high signal intensity as well as a high signal-to-noise ratio for all 32 genes. These results indicate that Phi29 v2 and OmniPlex WGA are useful methods for obtaining sufficient DNA from a limited amount of samples for the detection of microbes using microarrays.

摘要

使用微阵列对病原体进行基因诊断具有高通量检测的优势,但需要相对大量的DNA样本。为了获得足够量的DNA用于分子诊断,人们提出了几种全基因组扩增(WGA)方法。在本研究中,以土拉弗朗西斯菌和大肠杆菌为模型,我们比较了四种WGA方法在全基因组DNA扩增效率方面的差异,以便确定最适合制备用于微阵列分析的DNA的方法。使用四种方法从10 ng土拉弗朗西斯菌和大肠杆菌基因组DNA中均有可能获得超过1.5 μg的产物,但在多重置换扩增(MDA)或全基因组扩增试剂盒(OmniPlex WGA)中细菌基因扩增的偏差最小。当将扩增的DNA应用于由32种不同基因探针组成的微阵列载玻片时,MDA的Phi29 v2和OmniPlex WGA扩增的DNA对所有32个基因均显示出高信号强度以及高信噪比。这些结果表明,Phi29 v2和OmniPlex WGA是从有限量样本中获得足够DNA以使用微阵列检测微生物的有用方法。

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