Genotyping on a thermal gradient DNA chip
Genotyping on a thermal gradient DNA chip
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DOI：
10.1101/gr.790603
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发表时间：
2003-03-01
期刊：
影响因子：
7
通讯作者：
Fortina, P
中科院分区：
文献类型：
--
作者：
Kajiyama, T;Miyahara, Y;Fortina, P
具有离散、独立温度控制岛的硅基芯片已被开发用于 DNA 微阵列杂交研究。每个岛都包含一个由扩散层制成的加热器和一个基于 p-n 结的温度传感器，通过各向异性蚀刻在二氧化硅/氮化物表面上创建。随后将不同的反应基团添加到岛的表面，并将等位基因特异性寡核苷酸探针附着到芯片上的离散点上。使用通过 PCR 从基因组 DNA 衍生的 Cy5 标记的单链靶标进行杂交。通过测量杂交和洗涤后结合的染料标记靶标的荧光来评估结果。每个岛的温度可以设置为不同的值，以获得完美匹配和不匹配之间的最佳区别。这种方法有助于定义探针/目标退火的最佳温度，以及区分完美匹配与不匹配的固溶相目标。然后对热梯度 DNA 芯片进行基因分型测试，并给出了两个基因中四个不同位点的结果。对因子 VII 和血色素沉着病基因内的临床相关基因座实现了明确的分型。
Silicon-based chips with discrete, independently temperature-controlled islands have been developed for use in DNA microarray hybridization studies. Each island, containing a heater made of a diffusion layer and a temperature sensor based on a p-n junction, is created on a silicon dioxide/nitride surface by anisotropic etching. Different reactive groups are subsequently added to the surface of the islands, and allele-specific oligonucleotide probes are attached to discrete spots on the chip. Hybridization is performed with Cy5-tagged single-stranded targets derived by PCR from genomic DNA. Results are assessed by measuring fluorescence of bound dye-tagged targets after hybridization and washing. Temperatures at each island can be set at different values to obtain optimal distinction between perfect matches and mismatches. This approach facilitates definition of optimal temperatures for probe/target annealing and for distinction between perfectly matched versus mismatched solution-phase targets. The thermal gradient DNA chips were then tested for genotyping, and the results for four different loci in two genes are presented. Unambiguous typing was achieved for clinically relevant loci within the factor VII and hemochromatosis genes.