Toehold probe-based interrogation for haplotype phasing of long nucleic acid strands

Abstract

The arrangement of multiple single nucleotide polymorphisms (SNPs) in a gene, called a haplotype phase, is increasingly recognized as critical for accurate determination of disease risk and severity. However, conventional toehold-mediated strand displacement reactions are only able to interrogate SNPs, but not phase them since it is not known whether two SNPs in the same copy of the gene (cis) or in different copies of the same gene (trans) will give the same readout. While the rational introduction of an enzyme enables haplotype phasing, the complicated and stable secondary structure of long, single-stranded DNA sequences at room temperature limits its use. Complex nucleic acid structures make the hybridization of the probes difficult. Thus, we designed a molecular method to reveal the relative positions of SNPs located 1.4 kb apart in two copies of a gene by employing a competitive toehold probes and sink strategy at an elevated temperature. As such, we have successfully differentiated 20 nM of the 10 possible diplotypes in a long DNA target with two SNP sites located 1.4 kb apart within an hour without any additional amplification step. This offers a promising technology for accurate and fast haplotype phasing of SNPs that are over multiple kilobases away from each other.