Development of a Simple and Cost-Effective Method Based on T7 Endonuclease Cleavage for Detection of Single Nucleotide Polymorphisms

Aims: Single nucleotide polymorphisms (SNP) can be used as genetic markers and for risk assessment of allele-linked diseases, which can provide information for clinical diagnosis. Large-scale microarray and next-generation sequencing methods have made genome-wide SNP genotyping possible. However, in addition to their high cost, these techniques are dependent on having specialized equipment. Thus, there is a need for a simple genotyping method that can be implemented in a resource-limited environment. Methods: We developed a strategy for SNP genotyping based on T7 Endonuclease I cleavage and an enzyme-linked microparticle immune assay. Using this method, we genotyped two common SNP sites (rs11526468 and rs12979860). The quality of the genotyping process was validated. Results: Although a 70% false-negative rate was observed, no false-positive reactions were found. Therefore, multiple parallel repeat reactions can offset the possibility of mutation detection failure. Discussion: This method employs a duplicate reagent-dependent procedure, and therefore has the potential for integration into a portable kit for field utilization.