Development of DNA chips for ribosomal protein genes

在这项研究中，我们与爱沙尼亚塔尔图大学的metspalu博士合作开发了人类核糖体蛋白（RP）基因的DNA芯片，以检查RP基因缺陷在人类疾病中的可能参与。我们准备了以下两种DNA芯片；一个用于杂交，另一个用于引物延伸到芯片上。将40S核糖体小亚基蛋白cdna的RPex（核糖体蛋白基因表达）chipPCR产物（~ 250bp）固定在环氧硅化玻璃表面。用野生型HeLa细胞和热休克型HeLa细胞的总mRNA制备荧光标记DNA探针，杂交检测mRNA水平上的基因表达。虽然在这两种细胞中RP基因的表达没有显著差异，但我们在HeLa和胎盘rna的表达比较中发现了RPS17、RPS18、RPS24和RPS27的表达差异。提示RP基因表达水平的改变参与了癌变的发生。RPmu（核糖体蛋白基因突变）chipOligoncleotides （25 mer）特异的小亚基蛋白基因通过5'端氨基固定在环氧硅化玻璃表面。在将目标DNA与阵列杂交后，通过DNA聚合酶将目标依赖的寡核苷酸延伸用于将荧光标记的双脱氧终止物结合到引物中。超过80%的引物工作正常，阵列上出现了精确的单碱基扩展。这两种芯片将有助于研究RP基因在人类疾病中的缺陷或突变。

In this study, we have developed DNA chips for human ribosomal protein (RP) genes, in collaboration with Dr.Metspalu at Tartu University in Estonia, to examine the possible involvement of RP gene defects in human disorders. We prepared following two types of DNA chips ; one for hybridization and the other for primer extension on the chip.1. RPex (Ribosomal Protein gene expression) chipPCR products (〜250 bp) of cDNAs for 40S ribosomal small subunit proteins were immobilized onto an epoxy-silanized glass surface. The gene expression at the mRNA level was measured by hybridization of fluorescently labeled DNA probes prepared from total mRNA of either wild-type HeLa cells or heat-shock HeLa cells. Although there were no significant differences of the RP gene expression in these two cells, we found the differences in RPS17, RPS18, RPS24, and RPS27 when the expression was compared between the HeLa and placental RNAs. This suggests that the changes of the RP gene expression level are involved in the carcinogenesis.2. RPmu (Ribosomal Protein gene mutation) chipOligoncleotides (25 mer) specific to the small subunit protein genes were immobilized via 5'terminal amino group on an epoxy-silanized glass surface. After hybridization of target DNA to the array, target dependent oligonucleotide extension by a DNA polymerase is used to incorporate fluorescently labeled dideoxy terminators to the primers. More than 80% of the primers worked properly and precise single-base extensions were occurred on the array.These two chips shotuld be useful for studying the RP gene defects or mutations in human disorders.