Development and validation of Corynebacterium DNA microarrays
Development and validation of Corynebacterium DNA microarrays
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DOI：
10.1128/aem.67.5.2310-2318.2001
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发表时间：
2001-05-01
影响因子：
4.4
通讯作者：
Sinskey, AJ
中科院分区：
文献类型：
--
作者：
Loos, A;Glanemann, C;Sinskey, AJ
我们开发了DNA微阵列技术来研究谷氨酸棒状杆菌。用PCR扩增出52个C，谷氨酸基因编码的初代代谢酶，一式三份打印在载玻片上。总RNA是从C. glutamum发酵的指数生长和赖氨酸生产阶段收获的细胞中提取的。利用随机六聚体引物逆转录制备荧光标记的cdna，并将其杂交到微阵列上。为了建立一套该技术的基准指标，我们比较了同一载玻片上复制点之间的可变性，与同一标记反应的cdna杂交的载玻片之间的可变性，以及与不同标记反应制备的cdna杂交的载玻片之间的可变性。我们发现结果是可靠的和统计上可重复的。给定载玻片上重复点之间的点对点变异性为3.8%，单独载玻片上的点之间的点对点变异性为5.0%（尽管与相同的标记cDNA杂交），与来自单独逆转录反应的样品杂交的单独载玻片上的点之间的点对点变异性为8.1%，在所有条件下，平均点对点变异性为7.1%。此外，当我们检测两个发酵阶段之间基因表达的变化时，我们发现大多数基因的结果与使用其他方法观察到的结果一致。这些方法将为改进C，谷氨酸产生菌株的代谢工程工具箱提供有价值的补充。
We have developed DNA microarray techniques for studying Corynebacterium glutamicum. A set of 52 C, glutamicum genes encoding enzymes from primary metabolism was amplified by PCR and printed in triplicate onto glass slides. Total RNA was extracted from cells harvested during the exponential-growth and lysine production phases of a C. glutamicum fermentation. Fluorescently labeled CDNAs were prepared by reverse transcription using random hexamer primers and hybridized to the microarrays, To establish a set of benchmark metrics for this technique, we compared the variability between replicate spots on the same slide, between slides hybridized with cDNAs from the same labeling reaction, and between slides hybridized viith cDNAs prepared in separate labeling reactions. We found that the results were both robust and statistically reproducible. Spot-to-spot variability was 3.8% between replicate spots on a given slide, 5.0% between spots on separate slides (though hybridized with identical, labeled cDNA), and 8.1% between spots from separate slides hybridized with samples from separate reverse transcription reactions yielding an average spot to spot variability of 7.1% across all conditions. Furthermore, when we examined the changes in gene expression that occurred between the two phases of the fermentation, we found that results for the majority of the genes agreed with observations made using other methods, These procedures will be a valuable addition to the metabolic engineering toolbox for the improvement of C, glutamicum amino acid-producing strains.