Genotyping of multiple single nucleotide polymorphisms with hyperbranched rolling circle amplification and microarray

The emerging role of single nucleotide polymorphisms (SNPs) in clinical diagnostics and studies has created a need for simple and high-throughput genotyping methods. Previously, we developed a 3-dimensional polyacrylamide gel-based microarray (3-D microarray) of PCR-product. This method can detect single SNP locus from multiple DNA samples on one chip. Hyperbranched rolling circle amplification (HRCA) was used to recognize different SNP loci and amplify the fragments from genomic samples. Different HRCA products were used to fabricate the 3-D microarray, and dual-color fluorescent probes were used to detect signals. This assay was applied to genotype 2 SNP loci from a set of 6 genomic DNA samples on one chip. Universal acryl-modified primer and one pair of dual-color fluorescent probes were used for all sample detection to reduce the cost. We demonstrate that this assay can detect 10 ng genomic DNA. Combination of HRCA and 3-D microarray allows parallel discrimination of different alleles from different samples on a single chip. It is a feasible method for high-throughput mutation analysis and disease diagnosis.