Dufva Martin, Petersen Jesper, Stoltenborg Michael, Birgens Henrik, Christensen Claus B V
Microarray Group, Department of Micro and Nanotechnology, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark.
Anal Biochem. 2006 May 15;352(2):188-97. doi: 10.1016/j.ab.2006.03.008. Epub 2006 Mar 23.
Allele-specific hybridization to a DNA microarray can be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation to an agarose film grafted onto unmodified glass. Microarrays of TC-tagged probes immobilized on the agarose film can be used to diagnose mutations in the human beta-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding melting point temperature ( approximately 20 degrees C), a single stringency wash still gave sufficiently high discrimination signals between perfect match and mismatch probes to allow robust mutation detection. In all, 270 genotypings were performed on patient materials, and no genotype was incorrectly classified. Quality control experiments conducted using a target DNA specific for the TC tag of the immobilized probes showed that the spotting and hybridization procedure had a variance of 20%, indicating that signal differences as low as twofold could be detected between perfect match and mismatch. Together, our results show that the use of microarrays of TC-tagged probes that have been immobilized on agarose films grafted onto glass is a robust and inexpensive genotyping method.
等位基因特异性杂交至DNA微阵列可成为对患者DNA进行基因分型的一种有用方法。在本文中，我们证明，带有聚（T）10 - 聚（C）10尾（TC标签）但无其他修饰的13至17个碱基的寡核苷酸可通过紫外线照射交联至接枝于未修饰玻璃上的琼脂糖膜。固定在琼脂糖膜上的TC标签探针微阵列可用于诊断人类β-珠蛋白基因中的突变，该基因编码血红蛋白中的β链。尽管探针在熔点温度方面差异很大（约20℃），但单次严格洗涤仍能在完全匹配和错配探针之间给出足够高的区分信号，以实现可靠的突变检测。总共对患者材料进行了270次基因分型，没有基因型被错误分类。使用针对固定探针的TC标签的靶DNA进行的质量控制实验表明，点样和杂交程序的方差为20%，这表明在完全匹配和错配之间可检测到低至两倍的信号差异。总之，我们的结果表明，使用固定在接枝于玻璃上的琼脂糖膜上的TC标签探针微阵列是一种可靠且廉价的基因分型方法。
