Determinants of sensitivity and specificity in spotted DNA microarrays with unmodified oligonucleotides
Determinants of sensitivity and specificity in spotted DNA microarrays with unmodified oligonucleotides
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DOI：
10.1266/ggs.79.189
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发表时间：
2004-08-01
影响因子：
1.1
通讯作者：
Ishiura, M
中科院分区：
文献类型：
--
作者：
Kucho, KI;Yoneda, H;Ishiura, M
具有未修饰寡核苷酸的DNA微阵列是cDNA微阵列的具有成本效益的替代方案。本研究考察了寡核苷酸探针的纯度、长度、同源性和GC含量如何影响使用蓝藻基因的方法的灵敏度和特异性。省略了通过高压液相色谱法的寡核苷酸纯化，而没有显著降低杂交灵敏度。对于测试的三个基因中的两个，寡核苷酸长度的减少不会降低杂交灵敏度，并且用45 nt长的探针实现了最大灵敏度。与非靶DNA具有小于或等于71%连续序列同一性的寡核苷酸探针与序列的交叉杂交率<8%。随着GC含量从65%降低到55%或35%，交叉杂交减少。这些结果支持了选择产生敏感和特异性微阵列的未修饰寡核苷酸探针的以下标准：长度为45 nt，与非靶序列的同一性小于或等于71%，GC含量小于或等于55%。在大多数测试的细菌物种中，满足这些标准的寡核苷酸探针被成功地设计用于超过95%的基因。
DNA microarrays with unmodified oligonucleotides are a cost-effective alternative to cDNA microarrays. This study examined how purity, length, homology and GC content of the oligonucleotide probes influence the sensitivity and specificity of the method using cyanobacterial genes. Oligonucleotide purification by high pressure liquid chromatography was omitted without significant reduction in hybridization sensitivity. For two of three genes tested, a reduction in oligonucleotide length did not reduce hybridization sensitivity, and maximum sensitivity was achieved with probes that were 45 nt long. Oligonucleotide probes with less than or equal to71% contiguous sequence identity to non-target DNA cross-hybridized with the sequences at a rate of < 8%. Cross-hybridization decreased as the GC content decreased from 65% to 55% or 35%. These results support the following criteria for selecting unmodified oligonucleotide probes that generate sensitive and specific microarrays: length of 45 nt, less than or equal to71% identity to non-target sequences, and less than or equal to55% GC content. In most of the bacterial species tested, oligonucleotide probes meeting these criteria were successfully designed for more than 95% of genes.