Lindroos K, Liljedahl U, Raitio M, Syvänen A C
Molecular Medicine, Department of Medical Sciences, Uppsala University, 75185 Uppsala, Sweden.
Nucleic Acids Res. 2001 Jul 1;29(13):E69-9. doi: 10.1093/nar/29.13.e69.
In the microarray format of the minisequencing method multiple oligonucleotide primers immobilised on a glass surface are extended with fluorescent ddNTPs using a DNA polymerase. The method is a promising tool for large-scale single nucleotide polymorphism (SNP) detection. We have compared eight chemical methods for covalent immobilisation of the oligonucleotide primers on glass surfaces. We included both commercially available, activated slides and slides that were modified by ourselves. In the comparison the differently derivatised glass slides were evaluated with respect to background fluorescence, efficiency of attaching oligonucleotides and performance of the primer arrays in minisequencing reactions. We found that there are significant differences in background fluorescence levels among the different coatings, and that the attachment efficiency, which was measured indirectly using extension by terminal transferase, varied largely depending on which immobilisation strategy was used. We also found that the attachment chemistry affects the genotyping accuracy, when minisequencing on microarrays is used as the genotyping method. The best genotyping results were observed using mercaptosilane-coated slides attaching disulfide-modified oligonucleotides.
在微测序法的微阵列形式中，固定在玻璃表面的多个寡核苷酸引物利用DNA聚合酶与荧光双脱氧核苷酸三磷酸（ddNTPs）进行延伸。该方法是大规模单核苷酸多态性（SNP）检测的一种很有前景的工具。我们比较了将寡核苷酸引物共价固定在玻璃表面的八种化学方法。我们既包括市售的活化载玻片，也包括我们自己修饰的载玻片。在比较中，对不同衍生化的载玻片进行了背景荧光、寡核苷酸连接效率以及引物阵列在微测序反应中的性能等方面的评估。我们发现不同涂层之间的背景荧光水平存在显著差异，并且通过末端转移酶延伸间接测量的连接效率很大程度上取决于所使用的固定策略。我们还发现，当将微阵列上的微测序用作基因分型方法时，连接化学会影响基因分型的准确性。使用附着二硫键修饰寡核苷酸的巯基硅烷包被载玻片时，观察到了最佳的基因分型结果。
