Toehold probe-based interrogation for haplotype phasing of long nucleic acid strands. Issue 34 (12th August 2020)

• Record Type: Journal Article
• Authors: Zhuang, Xinyu; Lee Yu, Henson L.; Hsing, I-Ming
• Is Part Of: Analytical methods. Volume 12:Issue 34(2020)
• Page Start: 4185
• Page End: 4190
• Publication Date: 2020-08-12
• Subjects: Chemistry, Analytic -- Periodicals; Analytical biochemistry -- Periodicals; Chemical laboratories -- Standards -- Periodicals; 543.1905
• DOI: 10.1039/d0ay00946f
• Languages: English
• ISSNs: 1759-9660

Abstract

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. 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.