High-efficiency and high-fidelity ssDNA circularisation via the pairing of five 3′-terminal bases to assist LR-LAMP for the genotyping of single-nucleotide polymorphisms

The poor fidelity of T4 DNA ligase has always limited the simple detection of single-nucleotide polymorphisms (SNPs) and is only applicable to some special SNP types. This study developed a highly sensitive and specific detection method for SNPs based on high-fidelity single-stranded circularisation. It used T4 DNA ligase and rolling circle amplification (RCA) plus loop-mediated isothermal amplification (LAMP). Surprisingly, the cyclisation stage's efficiency greatly improved. The ligation fidelity was almost perfect via the unique pairing pattern between a long-paired base at the 5 ' terminus and only five bases at the 3 ' terminus on linear single-stranded DNA (l-DNA). Subsequently, LR-LAMP was performed and combined with the circularisation step for the simple detection of SNPs. The results showed that even 100 aM targets could be detected correctly and that a mutation rate of 0.1% or even 0.01% could be analysed via naked-eye visualisation or fluorescence detection, respectively. In addition, genomic DNA samples were used to evaluate the method, which indicated that it could effectively distinguish the SNPs of RPA190-T1145A in Phytophthora infestans. This strategy may play an important role in both circularisation of single-stranded DNA and detecting arbitrary SNPs.

Automated summary

A highly sensitive and specific SNP detection method was developed based on high-fidelity single-stranded circularisation. The efficiency of the cyclisation stage greatly improved, and the ligation fidelity was almost perfect. The method allowed for the simple detection of low mutation rate SNPs, and showed potential for various applications.