Oligonucleotide hybridization with magnetic separation assay for multiple SNP phasing

• Main Authors: Henson L. Lee Yu, Tsz Wing Fan, I-Ming Hsing
• Format: Article
• Language: English
• Published: Elsevier 2020-07-01
• Series: Analytica Chimica Acta: X
• Subjects: Multiple SNP Phasing; DNA probe Hybridization; Magnetic separation assay; DNA self-Assembly
• Online Access: http://www.sciencedirect.com/science/article/pii/S2590134620300128

Since humans have two copies of each gene, multiple mutations in different loci may or may not be found on the same strand of DNA (i.e., inherited from one parent). When a person is heterozygous at more than one position, the placement of these mutations, also called the haplotype phase, (i.e., cis for the same strand and trans for different strands) can result in the expression of different amount and type of proteins. In this work, we described an enzyme-free method to phase two single nucleotide polymorphisms (SNPs) using two fluorophore/quencher-labelled probes, where one of which was biotinylated. The fluorescence signal was obtained twice: first, after the addition of the labelled probes and second, after the addition of the magnetic beads. The first signal was shown to be proportional to the total number of SNP A and SNP B present in the target analyte, while the second signal showed a marked decrease of the fluorescence signal from the non-biotinylated probe when the SNPs were in trans, showing that the probe immobilized on the magnetic bead selectively captures targets with SNPs in a cis configuration. We then mimic the nature of the human genome which consists of two haplotype copies of each gene, and showed that 250 nM of the 10 possible pairs of haplotypes could be differentiated using a combination of fluorescence microscopy and fluorescence detection.