Analysis of single nucleotide polymorphism in human angiogenin using droplet-based microfluidics

Human angiogenin through its ribonucleolytic activity and nuclear translocation promotes angiogenesis and protects motor neurons from damage under stress conditions. Several heterozygous missense and loss of function mutations in the coding region of angiogenin have been implicated in the progression of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. Every year, thousands of new patients of ALS are diagnosed, making detection of angiogenin variants an urgent necessity. In this work, we report a method of SNP detection based on offline PCR amplification, of the target samples followed by bulk and droplet-based microfluidic fluorescence measurements. Our assay relied on the 5' nuclease-based reporter excision by Taq polymerase from two differently-labeled TaqMan minor groove binder (MGB) probes, one specific for the wild type and the other for K84E single nucleotide polymorphism (SNP). The reporter-specific fluorescence signals after 30 cycles of PCR from 20 ng starting template were determined, and the wild type and the K84E SNP fluorescence intensities were approximately 3.5-times and 6-times higher compared to the no template control reaction, respectively. The results on both bulk and droplet-based microfluidic fluorescence measurements are consistent with each other, and suggest that fluorescence detection using droplet-based microfluidics can be used for the detection of SNP in angiogenin.