Dinucleotide tag-based parallel reporter gene assay method enables efficient identification of regulatory mutations

The causal single nucleotide polymorphisms (SNPs) leading to increased cancer predisposition mainly function as gene regulatory elements, the evaluation of which largely relies on the parallel reporter gene assay system. However, the common DNA barcodes used in parallel reporter gene assay systems typically because nucleotide composition bias, and many barcodes must be allocated for each sequence to reduce the bias effect.

Here, a versatile dinucleotide-tag reporter system (DiR) that enables parallel analysis of regulatory elements with minimized bias based on next-generation sequencing is described. The DiR system is more robust than the classical luciferase assay method, particularly for the investigation of moderate-level regulatory elements. The authors applied the DiR-seq assay in the functional evaluation of SNPs with prostate cancer risk and nominated two and six regulatory SNPs in PC-3 and LNCaP cells, respectively.

The DiR system has great potential to advance the functional study of SNPs associated with polygenic disease risks.