Development of SNP and gene expression analysis method using DNA computing technology

建立了一种基于DNA计算的多重SNP分型和基因表达谱分析的方法，以及它们的直接模式分析方法。对于SNP分型分析，开发了使用Taq DNA连接酶的高度特异性编码反应和使用不对称PCR的同时扩增和解码反应。应用该方法对40个基因组DNA进行了28个SNPs的多重分型。样品这28个SNP是从人5号染色体上包括IL-4和IL-13基因的500 kb区域中随机选择的。该方法的成功率很高，因为除了一个单态SNP外，所有SNP的基因型都被成功确定。对于基因表达谱分析，开发了标准化编码方法以实现高度定量分析。由于编码效率不均匀，先前基于DNA计算的基因表达谱分析方法缺乏足够精确的cDNA定量。对100个具有均匀解链温度的DNA寡聚体的编码效率的实验揭示，效率与靶寡聚体的浓度成正比，而即使在均匀浓度下，效率也随靶而变化。基于这一事实，通过使用来自通过将等量的DNA寡聚体与靶基因特异性序列混合而制备的参考样品的信号，成功地消除了编码效率的不均匀性。将标准化编码方法应用于定量已知浓度范围为0.1 pM至10 pM的DNA寡聚体的混合物，以及从酵母和人培养细胞中提取的1 pg总RNA制备的cDNA。结果表明，该方法与qPCR方法相比具有更高的平行性和更高的准确性，与市售的DNA芯片和DNA芯片相比，其cDNA定量结果与qPCR结果的相关性更好。

A DNA-computing-based method for multiplex SNP typing and gene expression profiling and that for their direct pattern analysis have been developed. For a SNP typing analysis, a highly specific encoding reaction with Taq DNA ligase and a simultaneous amplification and decoding reaction using asymmetric PCR were developed. The method was applied to the analysis of multiplex typing for 28 SNPs using 40 genomic DNA. samples. The 28 SNPs were randomly selected from a 500-kb region including the IL-4 and IL-13 genes on human chromosome 5. The method gave a high success rate because the genotypes of all SNPs but one monomorphic SNP were successfully determined. For a gene expression profiling analysis, a normalized-encoding method was developed to realize a highly quantitative analysis The previous DNA-computing-based method for gene expression profiling was lacking in sufficiently precise quantification of cDNA due to the non-uniform encoding efficiency. Experiments on the encoding efficiency of one hundred DNAoligomers with a uniform melting temperature disclosed that the efficiency was proportional to the concentration of the target oligomer while it varied with the target even at a uniform concentration. Based on this fact, the non-uniformity of the encoding efficiency was successfully cancelled by using the signal from the reference sample prepared by mixing equal amounts of DNAoligomers with target gene specific sequences. The normalized-encoding method was applied to quantification of a mixture of DNAoligomers of known concentrations ranging from 0.1 pM to 10 pM, and that of cDNAs prepared from 1 pg of total RNAextracted from yeast and human culture cells. The method succeeded in quantification that is as precise as qPCR and more parallel than it The results of cDNAquantification by the method gave better correlations with those by qPCR than commercially available DNAmicroarrays and DNAchips..