Franke-Whittle Ingrid H, Klammer Susanne H, Mayrhofer Sabine, Insam Heribert
Institute for Microbiology, University of Innsbruck, Technikerstrasse 25d, A-6020 Innsbruck, Austria.
J Microbiol Methods. 2006 Apr;65(1):117-26. doi: 10.1016/j.mimet.2005.06.011. Epub 2005 Jul 25.
Different labeling methods were studied to compare various approaches to the preparation of labeled target DNA for microarray experiments. The methods under investigation included a post-PCR labeling method using the Klenow fragment and a DecaLabel DNA labeling kit, the use of a Cy3-labeled forward primer in the PCR, generating either double-stranded or single-stranded PCR products, and the incorporation of Cy3-labeled dCTPs in the PCR. A microarray that had already been designed and used for the detection of microorganisms in compost was used in the study. PCR products from the organisms Burkholderia cepacia and Staphylococcus aureus were used in the comparison study, and the signals from the probes for these organisms analyzed. The highest signals were obtained when using the post-PCR labeling method, although with this method, more non-specific hybridizations were found. Single-stranded PCR products that had been labeled by the incorporation of a Cy3-labeled forward primer in the PCR were found to give the next highest signals upon hybridization for a majority of the tested probes, with less non-specific hybridizations. Hybridization with double-stranded PCR product labeled with a Cy3-labeled forward primer, or labeled by the incorporation of Cy3-labeled dCTPs resulted in acceptable signal to noise ratios for all probes except the UNIV 1389a and Burkholderia genus probes, both located toward the 3' end of the 16S rRNA gene. The comparison of the different DNA labeling methods revealed that labeling via the Cy3-forward primer approach is the most appropriate of the studied methods for the preparation of labeled target DNA for our purposes.
研究了不同的标记方法，以比较用于微阵列实验的标记靶DNA制备的各种方法。所研究的方法包括使用Klenow片段和DecaLabel DNA标记试剂盒的PCR后标记方法、在PCR中使用Cy3标记的正向引物（产生双链或单链PCR产物）以及在PCR中掺入Cy3标记的dCTP。本研究使用了一个已经设计好并用于检测堆肥中微生物的微阵列。来自洋葱伯克霍尔德菌和金黄色葡萄球菌的PCR产物用于比较研究，并分析这些微生物探针的信号。使用PCR后标记方法时获得了最高信号，不过用这种方法发现了更多的非特异性杂交。在PCR中通过掺入Cy3标记的正向引物标记的单链PCR产物，在大多数测试探针杂交时产生的信号次之，且非特异性杂交较少。用Cy3标记的正向引物标记的双链PCR产物或通过掺入Cy3标记的dCTP标记的双链PCR产物进行杂交，除了位于16S rRNA基因3'端的UNIV 1389a和伯克霍尔德菌属探针外，所有探针的信噪比均可接受。不同DNA标记方法的比较表明，对于我们的目的而言，通过Cy3正向引物方法进行标记是制备标记靶DNA的研究方法中最合适的。
