Marking of specific sequences in double-stranded DNA molecules--SNP detection and direct observation

• Published in: Genome research 2004-12, Vol.14 (12), p.2478-2485
• Main Authors: Shigemori, Yasushi, Haruta, Hirotaka, Okada, Takao, Oishi, Michio
• Format: Article
• Language: English
• Subjects: Blotting, Southern; DNA - genetics; DNA - metabolism; DNA Ligases - metabolism; DNA Primers; Methods; Microscopy, Atomic Force; Nucleic Acid Hybridization - genetics; Oligonucleotides - metabolism; Polymorphism, Single Nucleotide - genetics; Rec A Recombinases - metabolism
• ISSN: 1088-9051; 1549-5469
• DOI: 10.1101/gr.2789604

In this study, we describe a simple method to mark specific sequences in double-stranded DNA molecules. For the marking, we used two specifically designed oligonucleotides, one of which is complementary to the sequence to be marked and the other, serving as a splint, to make the marking stable and detectable by subsequent various analytical means. In the presence of the two deoxyoligonucleotides, whereas RecA protein-mediated reaction converts the sequence to be marked to a regional triple-stranded structure with the complementary (probing) oligonucleotide, DNA ligase transforms it to a stable multi- (possibly quintuple) stranded structure with the splint oligonucleotide. The whole marking process is simple and completed in a single reaction mixture. Because RecA protein makes the marking to proceed with high fidelity, we were able to mark (detect) SNPs in complex genomes like human's. Furthermore, the structure of the marked sequence is stable and quite distinct enough to be readily detectable by biochemical means or direct observation by scanning probe microscopy.