Dissociation analysis in polymerase chain reaction and 1x SSC buffer as a prerequisite for selection of 13mer microarray probe sets with uniform hybridization behavior
Dissociation analysis in polymerase chain reaction and 1x SSC buffer as a prerequisite for selection of 13mer microarray probe sets with uniform hybridization behavior
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DOI：
10.1385/mb:29:1:01
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发表时间：
2005-01-01
影响因子：
2.6
通讯作者：
Frey, JE
中科院分区：
文献类型：
--
作者：
Pfunder, M;Frey, JE
均匀的探针杂交是微阵列稳健设计的先决条件。开发了精心设计的算法以选择具有均匀解链温度（Tm）的探针组。然而，至少短寡核苷酸（< 20 bp）显示芯片上杂交效率的大变化，即使用最先进的算法设计。这种变化可以解释的立体效应和固体表面上的干扰，以及由化学条件，可能会偏离用于开发的algorithms.We设计了412随机13聚体双链体的条件，研究最近邻算法的Tm和Tm值之间的差异测定的聚合酶链反应（PCR）和1X SSC缓冲液中的解离分析。我们测试了理论Tin值与经验Tin值对芯片上40个双链体的杂交变化的影响。尽管经验方法导致杂交效率的预测稍好，但所观察到的变化中只有不到五分之一（17%）可以单独用因子Tm解释。我们的结论是，国家的最先进的算法可以用于短的寡核苷酸探针的第一选择，但它是必要的，然后进行芯片上的选择，以获得一个统一的杂交行为的探针集。
Homogeneous probe hybridization is a prerequisite for the robust design of microarrays. Elaborate algorithms were developed to select for probe sets with uniform melting temperatures (Tm). However, at least short oligonucleotides (< 20 bp) show large variation in the on-chip hybridization efficiency even if designed with state-of-the-art algorithms. This variation can be explained by steric effects and interferences on the solid surface as well as by chemical conditions that may deviate from conditions used to develop the algorithms.We designed 412 random 13mer duplexes to study the differences between the Tm of nearest-neighbor algorithms and the Tm values measured by dissociation analysis in polymerase chain reaction (PCR) and 1X SSC buffer. We tested the effects of theoretical vs empirical Tin values on the hybridization variation of 40 duplexes on-chip. Although the empirical approach resulted in a slightly better prediction of hybridization efficiency, less than one-fifth (17%) of the observed variation could be explained by the factor Tm alone. We conclude that state-of-the-art algorithms can be used for a first selection of short oligonucleotide probes, but that it is then necessary to perform an on-chip selection to obtain a probe set with a uniform hybridization behavior.