Lindroos Katarina, Sigurdsson Snaevar, Johansson Karin, Rönnblom Lars, Syvänen Ann-Christine
Department of Medical Sciences, Uppsala University, 75185 Uppsala, Sweden.
Nucleic Acids Res. 2002 Jul 15;30(14):e70. doi: 10.1093/nar/gnf069.
We selected 125 candidate single nucleotide polymorphisms (SNPs) in genes belonging to the human type 1 interferon (IFN) gene family and the genes coding for proteins in the main type 1 IFN signalling pathway by screening databases and by in silico comparison of DNA sequences. Using quantitative analysis of pooled DNA samples by solid-phase mini-sequencing, we found that only 20% of the candidate SNPs were polymorphic in the Finnish and Swedish populations. To allow more effective validation of candidate SNPs, we developed a four-colour microarray-based mini-sequencing assay for multiplex, quantitative allele frequency determination in pooled DNA samples. We used cyclic mini-sequencing reactions with primers carrying 5'-tag sequences, followed by capture of the products on microarrays by hybridisation to complementary tag oligonucleotides. Standard curves prepared from mixtures of known amounts of SNP alleles demonstrate the applicability of the system to quantitative analysis, and showed that for about half of the tested SNPs the limit of detection for the minority allele was below 5%. The microarray-based genotyping system established here is universally applicable for genotyping and quantification of any SNP, and the validated system for SNPs in type 1 IFN-related genes should find many applications in genetic studies of this important immunoregulatory pathway.
我们通过筛选数据库以及对DNA序列进行电子比较，在属于人类1型干扰素（IFN）基因家族的基因以及主要1型IFN信号通路中编码蛋白质的基因中，挑选出了125个候选单核苷酸多态性（SNP）。通过对混合DNA样本进行固相微测序定量分析，我们发现，在芬兰和瑞典人群中，只有20%的候选SNP具有多态性。为了更有效地验证候选SNP，我们开发了一种基于四色微阵列的微测序检测方法，用于在混合DNA样本中进行多重定量等位基因频率测定。我们使用携带5'-标签序列的引物进行循环微测序反应，随后通过与互补标签寡核苷酸杂交将产物捕获在微阵列上。由已知量的SNP等位基因混合物制备的标准曲线证明了该系统在定量分析中的适用性，并表明对于大约一半的测试SNP，次要等位基因的检测限低于5%。这里建立的基于微阵列的基因分型系统普遍适用于任何SNP的基因分型和定量，并且经过验证的1型IFN相关基因SNP系统应该会在这一重要免疫调节途径的遗传研究中得到广泛应用。
