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

点击翻译按钮获取中文摘要

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..