Revision of the nonequilibrium thermal dissociation and stringent washing approaches for identification of mixed nucleic acid targets by microarrays.
Revision of the nonequilibrium thermal dissociation and stringent washing approaches for identification of mixed nucleic acid targets by microarrays.
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DOI：
10.1093/nar/gkm154
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发表时间：
2007
影响因子：
14.9
通讯作者：
Noble PA
中科院分区：
文献类型：
--
作者：
Pozhitkov AE;Stedtfeld RD;Hashsham SA;Noble PA
微阵列实验通常涉及去除杂化非特异性靶标的洗涤步骤，目的是提高信噪比。洗涤的质量最终影响芯片的下游分析和解释。缺乏旨在理解微阵列混合目标解离的基础研究使得开发有意义的洗涤/解离方案变得困难。为了填补这一空白，我们检测了47对完全匹配（PM）和双失配（MM）双相的活化能和指数前系数，发现PM和MM双相的动力学没有统计学差异。基于这些发现，我们评估了非平衡热解离（NTD）方法，该方法已被用于识别混合目标样品中的特定微生物目标。我们发现，各种洗涤方案和NTD方法的主要前提可能会受到严重损害，因为：(i)非特异性双链并不总是在特异性双链之前解离，（ii） PM和MM双链的解离率之间的关系取决于温度和双链顺序。特别是对于NTD，我们表明先前建议使用参考曲线、曲线指数和温度梯度会导致错误的结论。
Microarray experiments typically involve washing steps that remove hybridized nonspecific targets with the purpose of improving the signal-to-noise ratio. The quality of washing ultimately affects downstream analysis of the microarray and interpretation. The paucity of fundamental studies directed towards understanding the dissociation of mixed targets from microarrays makes the development of meaningful washing/dissociation protocols difficult. To fill the void, we examined activation energies and preexponential coefficients of 47 perfect match (PM) and double-mismatch (MM) duplex pairs to discover that there was no statistical difference between the kinetics of the PM and MM duplexes. Based on these findings, we evaluated the nonequilibrium thermal dissociation (NTD) approach, which has been used to identify specific microbial targets in mixed target samples. We found that the major premises for various washing protocols and the NTD approach might be seriously compromised because: (i) nonspecific duplexes do not always dissociate before specific ones, and (ii) the relationship between dissociation rates of the PM and MM duplexes depends on temperature and duplex sequence. Specifically for the NTD, we show that previously suggested use of reference curves, indices of curves and temperature ramps lead to erroneous conclusions.
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影响因子：
2.9
作者：
MARKY, LA;BRESLAUER, KJ
通讯作者：
BRESLAUER, KJ
影响因子：
14.9
作者：
IKUTA, S;TAKAGI, K;ITAKURA, K
通讯作者：
ITAKURA, K
影响因子：
14.9
作者：
KOZYAVKIN, SA;LYUBCHENKO, YL
通讯作者：
LYUBCHENKO, YL
影响因子：
5.1
作者：
Li, ESY;Ng, JKK;Liu, WT
通讯作者：
Liu, WT
影响因子：
4.4
作者：
Loy, A;Lehner, A;Wagner, M
通讯作者：
Wagner, M