Experimental optimization of probe length to increase the sequence specificity of high-density oligonucleotide microarrays.
Experimental optimization of probe length to increase the sequence specificity of high-density oligonucleotide microarrays.
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DOI：
10.1186/1471-2164-8-373
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发表时间：
2007-10-16
期刊：
影响因子：
4.4
通讯作者：
Yomo T
中科院分区：
文献类型：
--
作者：
Suzuki S;Ono N;Furusawa C;Kashiwagi A;Yomo T
高密度寡核苷酸芯片广泛用于全基因组表达和遗传变异的分析。Affymetrix GeneChips -常见的高密度寡核苷酸阵列-包含通过改变PM的单个核苷酸生成的完全匹配（PM）和错配（MM）探针，以估计交叉杂交。然而，当PM和MM探针之间的靶特异性杂交量的差异小于交叉杂交量的方差时，一部分MM探针表现出比PM更大的信号强度。因此，对于精确分析而言，需要对单核苷酸错配具有更高特异性的PM和MM探针对。为了研究单核苷酸错配的特异性，我们设计了一个定制的阵列，将不同长度（14- 25-mer）的探针连接到阵列的表面和所有可能的单核苷酸错配，并将人工合成的25-mer寡脱氧核糖核苷酸作为目标在本体溶液中杂交，以避免交叉杂交的影响。结果表明，随着探针长度的增加，靶分子的可用性有限。由于这种效应，较长探针的序列特异性降低，即使在转录组分析的通常背景条件下也证实了这一点。我们的研究表明，特异性的最佳探针长度为19-21-mer。这一结论将有助于改进基因芯片的设计，用于转录组分析和突变筛查。
High-density oligonucleotide arrays are widely used for analysis of genome-wide expression and genetic variation. Affymetrix GeneChips – common high-density oligonucleotide arrays – contain perfect match (PM) and mismatch (MM) probes generated by changing a single nucleotide of the PMs, to estimate cross-hybridization. However, a fraction of MM probes exhibit larger signal intensities than PMs, when the difference in the amount of target specific hybridization between PM and MM probes is smaller than the variance in the amount of cross-hybridization. Thus, pairs of PM and MM probes with greater specificity for single nucleotide mismatches are desirable for accurate analysis. To investigate the specificity for single nucleotide mismatches, we designed a custom array with probes of different length (14- to 25-mer) tethered to the surface of the array and all possible single nucleotide mismatches, and hybridized artificially synthesized 25-mer oligodeoxyribonucleotides as targets in bulk solution to avoid the effects of cross-hybridization. The results indicated the finite availability of target molecules as the probe length increases. Due to this effect, the sequence specificity of the longer probes decreases, and this was also confirmed even under the usual background conditions for transcriptome analysis. Our study suggests that the optimal probe length for specificity is 19–21-mer. This conclusion will assist in improvement of microarray design for both transcriptome analysis and mutation screening.
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DOI：
10.1073/pnas.1534744100
发表时间：
2003-09-30
影响因子：
11.1
作者：
Mei, R;Hubbell, E;Webster, TA
通讯作者：
Webster, TA
DOI：
10.1073/pnas.011404098
发表时间：
2001-01-02
影响因子：
11.1
作者：
Li, C;Wong, WH
通讯作者：
Wong, WH
影响因子：
5.1
作者：
Li, ESY;Ng, JKK;Liu, WT
通讯作者：
Liu, WT
影响因子：
2.4
作者：
Naef, F;Lim, DA;Magnasco, M
通讯作者：
Magnasco, M
影响因子：
2.1
作者：
Irizarry, RA;Hobbs, B;Speed, TP
通讯作者：
Speed, TP