Calabretta Alessandro, Tedeschi Tullia, Di Cola Gabriella, Corradini Roberto, Sforza Stefano, Marchelli Rosangela
Department of Organic and Industrial Chemistry, University of Parma, Viale G.P. Usberti 17a, I-43100, Parma, Italy.
Mol Biosyst. 2009 Nov;5(11):1323-30. doi: 10.1039/b909912n. Epub 2009 Aug 24.
Four modified PNAs containing one chiral monomer bearing two arginine-derived side chains, with the correct configuration for specific and stable DNA binding, were synthesized, complementary to two DNA tracts in the APOE gene containing SNPs related to the insurgence of Alzheimer's disease. PNA binding performances were first tested in solution against complementary and mismatched oligonucleotides by measuring melting temperatures, and showed high specificity in SNP recognition. In order to set up a new diagnostic platform for APOE genotyping, PNA microarrays were then developed with the synthesized modified PNAs. PNA probe deposition protocols on microarrays were optimized in order to minimize cross-contamination due to carry over. The microarrays obtained by arginine-based PNA deposition were incubated with complementary and mismatched oligonucleotides, showing excellent mismatch recognition on the microarray platform. The specificity of the microarrays was finally tested with oligonucleotide mixtures simulating the real genotype profiles. Six different hybridisation patterns related to six different genotypes in the APOE gene were found to be clearly distinct in microarray experiments, demonstrating the potential of this approach for highly specific genetic analysis.
合成了四种修饰的肽核酸（PNA），它们含有一个带有两个精氨酸衍生侧链的手性单体，具有正确的构型以实现特异性和稳定的DNA结合，与载脂蛋白E（APOE）基因中两个含有与阿尔茨海默病发生相关单核苷酸多态性（SNP）的DNA片段互补。首先通过测量解链温度在溶液中针对互补和错配的寡核苷酸测试PNA的结合性能，结果显示其在SNP识别方面具有高特异性。为了建立一个用于APOE基因分型的新诊断平台，随后用合成的修饰PNA开发了PNA微阵列。对微阵列上PNA探针的沉积方案进行了优化，以尽量减少由于残留导致的交叉污染。将通过基于精氨酸的PNA沉积获得的微阵列与互补和错配的寡核苷酸一起孵育，结果显示在微阵列平台上具有出色的错配识别能力。最后用模拟真实基因型谱的寡核苷酸混合物测试微阵列的特异性。在微阵列实验中发现，与APOE基因中六种不同基因型相关的六种不同杂交模式明显不同，证明了这种方法用于高特异性基因分析的潜力。
