Department of Micro and Nanotechnology, DTU Nanotech, Technical University of Denmark, Lyngby, Denmark.
PLoS One. 2011 Mar 22;6(3):e14777. doi: 10.1371/journal.pone.0014777.
The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions. These regions include large variations in G+C content, and structural features like hairpins.
METHODS/FINDINGS: We describe a rational, stable method for screening and combining assay conditions for the genetic analysis of 42 Phenylketonuria-associated mutations in the phenylalanine hydroxylase gene. The mutations are located in regions with large variations in G+C content (20-75%). Custom-made microarrays with different lengths of complementary probe sequences and spacers were hybridized with pooled PCR products of 12 exons from each of 38 individual patient DNA samples. The arrays were washed with eight buffers with different stringencies in a custom-made microfluidic system. The data were used to assess which parameters play significant roles in assay development.
Several assay development methods found suitable probes and assay conditions for a functional test for all investigated mutation sites. Probe length, probe spacer length, and assay stringency sufficed as variable parameters in the search for a functional multiplex assay. We discuss the optimal assay development methods for several different scenarios.
基于微阵列的已知突变引起的疾病的基因检测的发展变得越来越重要。开发功能基因分型检测的关键障碍是无论这些突变位于基因组区域的何处，都需要对其进行基因分型。这些区域包括 G+C 含量的大变化，以及发夹等结构特征。
方法/结果：我们描述了一种合理、稳定的方法，用于筛选和组合苯丙氨酸羟化酶基因中 42 个苯丙酮尿症相关突变的遗传分析。这些突变位于 G+C 含量（20-75%）变化较大的区域。使用定制的微流控系统，用不同长度的互补探针序列和间隔物定制的微阵列与来自 38 个个体患者 DNA 样本的每个 12 个外显子的聚合酶链反应产物进行杂交。将阵列用 8 种具有不同严格性的缓冲液在定制的微流体系统中洗涤。该数据用于评估哪些参数在检测开发中起重要作用。
几种检测开发方法为所有研究的突变位点找到了适合功能测试的合适探针和检测条件。探针长度、探针间隔物长度和检测严格性足以作为寻找功能多重检测的变量参数。我们讨论了几种不同情况下的最佳检测开发方法。
