Landi Stefano, Gemignani Federica, Gioia-Patricola Lydie, Chabrier Amélie, Canzian Federico
International Agency for Research on Cancer, Lyon, France.
Biotechniques. 2003 Oct;35(4):816-20, 822, 824-7. doi: 10.2144/03354mt03.
We present an oligonucleotide microarray ("MetaboChip") based on the arrayed primer extension (APEX) technique, allowing genotyping of single nucleotide polymorphisms (SNPs) in genes of interest for cancer susceptibility and pharmacogenetics. APEX consists of a sequencing reaction primed by an oligonucleotide anchored with its 5' end to a glass slide and terminating one nucleotide before the polymorphic site. The extension with one fluorescently labeled dideoxynucleotide complementary to the template reveals the polymorphism. Ninety-three SNPs in 42 genes were selected among those resequenced in the context of the SNP500 project, using a set of 102 reference DNA samples from the Coriell Biorepository. Selected SNPs belong to the following genes: ADH1B, ALDH2, APEX, CDKN2A, COMT, CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C19, CYP2C9, CYP2E1, CYP3A4, DRD2, DRD4, EPHX1, ERCC1, ERCC2, ERCC4, ERCC5, GRPR, GSTA4, GSTM3, GSTP1, GSTT2, LIG3, MDM2, MGMT, MPO, NAT1, NAT2, NQO1, OGG1, PCNA, POLB, SLC6A3, SOD2, TP53, XRCC1, XRCC2, XRCC3, and XRCC9. We assessed the performance of APEX by comparing the results obtained with MetaboChip against those reported by the SNP500. Among 88 SNPs that yielded signals, 6 showed less than 99% of concordance, whereas 82 performed accurately, showing that APEX is a reliable and sensitive genotyping method.
我们展示了一种基于引物延伸（APEX）技术的寡核苷酸微阵列（“代谢芯片”），可对癌症易感性和药物遗传学相关的感兴趣基因中的单核苷酸多态性（SNP）进行基因分型。APEX由一个测序反应组成，该反应由一个寡核苷酸引发，其5'端锚定在载玻片上，并在多态性位点前一个核苷酸处终止。用一种与模板互补的荧光标记双脱氧核苷酸进行延伸，从而揭示多态性。在SNP500项目背景下重新测序的基因中，使用来自科里尔生物样本库的102个参考DNA样本，从42个基因中选择了93个SNP。所选的SNP属于以下基因：ADH1B、ALDH2、APEX、CDKN2A、COMT、CYP1A1、CYP1A2、CYP1B1、CYP2A6、CYP2C19、CYP2C9、CYP2E1、CYP3A4、DRD2、DRD4、EPHX1、ERCC1、ERCC2、ERCC4、ERCC5、GRPR、GSTA4、GSTM3、GSTP1、GSTT2、LIG3、MDM2、MGMT、MPO、NAT1、NAT2、NQO1、OGG1、PCNA、POLB、SLC6A3、SOD2、TP53、XRCC1、XRCC2、XRCC3和XRCC9。我们通过比较代谢芯片获得的结果与SNP500报告的结果，评估了APEX的性能。在产生信号的88个SNP中，6个的一致性低于99%，而82个表现准确，表明APEX是一种可靠且灵敏的基因分型方法。
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