Two-Tailed PCR for Precision Diagnostics

Two-Tailed PCR for Precision Diagnostics

Abstract
We present Two-Tailed PCR (2T-PCR), which is a new exceedingly sequence specific and sensitive method to analyse short nucleic acids and rare sequence variant analyses. 2T-PCR takes advantage of a target-specific primer composed of two hemiprobes, complementary to two different parts of the target molecule, connected by a hairpin structure. The introduction of short hemiprobes that sense the target sequence confers exceeding specificity while maintaining the very high sensitivity of PCR. Highly similar targets can be distinguished with superior precision irrespectively of the position of the variant nucleotide. The target molecule can be as short as some 15 bases, making 2T-PCR the preferred method for analyzing fragmented nucleic acids such as cell-free DNA, forensic samples, ancient DNA, formalin fixed and paraffin embedded (FFPE) material, as well as naturally short nucleic acids such as microRNAs. 2T-PCR is readily multiplexed and is particularly suitable for digital PCR.