Optimized detection of sequence variation in heterozygous genomes using DNA microarrays with isothermal-melting probes
Optimized detection of sequence variation in heterozygous genomes using DNA microarrays with isothermal-melting probes
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DOI：
10.1073/pnas.0913883107
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发表时间：
2010-01-26
影响因子：
11.1
通讯作者：
Botstein, David
中科院分区：
文献类型：
--
作者：
Gresham, David;Curry, Bo;Botstein, David
使用DNA微阵列来识别核苷酸变异几乎有20年的历史。探针设计和实验条件的各种改进使该技术达到了可以在未混合的样品中有效检测单核苷酸差异的程度，尽管开发了用于检测混合序列的可靠方法（例如，例如，在一个实施例中，杂合子）仍然具有挑战性。令人惊讶的是，探针设计参数和实验条件的全面研究，优化单核苷酸多态性（SNP）的歧视还没有被报道，所以这种技术的局限性仍然不确定。通过靶向两种酿酒酵母菌株之间不同的24，549个SNP，我们研究了SNP在不同杂交条件下对不同长度的DNA微阵列探针的杂交效率的影响。我们发现，优化序列鉴别的关键参数是探针解链温度（T-m）和进行杂交反应的温度之间的关系。通过改变探针的长度，可以设计含有相同Tm探针的微阵列来利用这种关系。我们证明，使用这样的微阵列，我们可以使用SNPScanner算法检测到>90%的纯合SNP和>80%的杂合SNP。在这项研究中确定的优化设计和实验参数应指导DNA微阵列设计的应用程序，需要序列的歧视，如突变检测，未混合和混合样品的基因分型，和等位基因特异性基因表达。此外，设计具有对错配的优化灵敏度的微阵列探针应该增加标准微阵列应用的准确性，例如拷贝数变异检测和基因表达分析。
The use of DNA microarrays to identify nucleotide variation is almost 20 years old. A variety of improvements in probe design and experimental conditions have brought this technology to the point that single-nucleotide differences can be efficiently detected in unmixed samples, although developing reliable methods for detection of mixed sequences (e. g., heterozygotes) remains challenging. Surprisingly, a comprehensive study of the probe design parameters and experimental conditions that optimize discrimination of single-nucleotide polymorphisms (SNPs) has yet to be reported, so the limits of this technology remain uncertain. By targeting 24,549 SNPs that differ between two Saccharomyces cerevisiae strains, we studied the effect of SNPs on hybridization efficiency to DNA microarray probes of different lengths under different hybridization conditions. We found that the critical parameter for optimization of sequence discrimination is the relationship between probe melting temperature (T-m) and the temperature at which the hybridization reaction is performed. This relationship can be exploited through the design of microarrays containing probes of equal T-m by varying the length of probes. We demonstrate using such a microarray that we detect >90% homozygous SNPs and >80% heterozygous SNPs using the SNPScanner algorithm. The optimized design and experimental parameters determined in this study should guide DNA microarray designs for applications that require sequence discrimination such as mutation detection, genotyping of unmixed and mixed samples, and allele-specific gene expression. Moreover, designing microarray probes with optimized sensitivity to mismatches should increase the accuracy of standard microarray applications such as copy-number variation detection and gene expression analysis.