Zabarovsky Eugene R, Petrenko Lev, Protopopov Alexei, Vorontsova Olga, Kutsenko Alexey S, Zhao Yanyan, Kilosanidze Gelena, Zabarovska Veronika, Rakhmanaliev Elian, Pettersson Bertil, Kashuba Vladimir I, Ljungqvist Olle, Norin Elisabeth, Midtvedt Tore, Möllby Roland, Winberg Gösta, Ernberg Ingemar
Microbiology and Tumor Biology Center, Department of Cell and Molecular Biology, Karolinska Institute, 171 77 Stockholm, Sweden.
Nucleic Acids Res. 2003 Aug 15;31(16):e95. doi: 10.1093/nar/gng096.
We have developed a new type of microarray, restriction site tagged (RST), for example NotI, microarrays. In this approach only sequences surrounding specific restriction sites (i.e. NotI linking clones) were used for generating microarrays. DNA was labeled using a new procedure, NotI representation, where only sequences surrounding NotI sites were labeled. Due to these modifications, the sensitivity of RST microarrays increases several hundred-fold compared to that of ordinary genomic microarrays. In a pilot experiment we have produced NotI microarrays from Gram-positive and Gram-negative bacteria and have shown that even closely related Escherichia coli strains can be easily discriminated using this technique. For example, two E.coli strains, K12 and R2, differ by less than 0.1% in their 16S rRNA sequences and thus the 16S rRNA sequence would not easily discriminate between these strains. However, these strains showed distinctly different hybridization patterns with NotI microarrays. The same technique can be adapted to other restriction enzymes as well. This type of microarray opens the possibility not only for studies of the normal flora of the gut but also for any problem where quantitative and qualitative analysis of microbial (or large viral) genomes is needed.
我们已经开发出一种新型微阵列，即限制性位点标记（RST）微阵列，例如NotI微阵列。在这种方法中，仅使用特定限制性位点周围的序列（即NotI连接克隆）来生成微阵列。DNA使用一种新方法进行标记，即NotI代表性标记，其中仅对NotI位点周围的序列进行标记。由于这些改进，RST微阵列的灵敏度比普通基因组微阵列提高了数百倍。在一项初步实验中，我们从革兰氏阳性菌和革兰氏阴性菌制备了NotI微阵列，并表明使用该技术甚至可以轻松区分密切相关的大肠杆菌菌株。例如，两种大肠杆菌菌株K12和R2，它们的16S rRNA序列差异小于0.1%，因此16S rRNA序列不容易区分这些菌株。然而，这些菌株在NotI微阵列上显示出明显不同的杂交模式。同样的技术也可以适用于其他限制性酶。这种类型的微阵列不仅为肠道正常菌群的研究开辟了可能性，也为任何需要对微生物（或大型病毒）基因组进行定量和定性分析的问题提供了可能。
