NBD‐Based Green Fluorescent Ligands for Typing of Thymine‐Related SNPs by Using an Abasic Site‐Containing Probe DNA |
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| Authors: | Viruthachalam Thiagarajan Arivazhagan Rajendran Hiroyuki Satake Seiichi Nishizawa Norio Teramae |
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| Affiliation: | 1. Department of Chemistry, Graduate School of Science, Tohoku University, Aoba‐ku, Sendai 980‐8578 (Japan), Fax: (+81)?22‐795‐6552;2. CREST (Japan) Science and Technology Agency (JST), Aoba‐ku, Sendai 980‐8578 (Japan);3. Present address: CEA, Institut de Biologie et Technologies de Saclay (IBITECS) and CNRS, Gif‐sur‐Yvette, 91191 (France);4. Present address: Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 7‐1‐20 Minatojima‐minamimachi, Chuo‐Ku, Kobe 650‐0047 (Japan) |
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| Abstract: | The binding behavior of green fluorescent ligands, derivatives of 7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD), with DNA duplexes containing an abasic (AP) site is studied by thermal denaturation and fluorescence experiments. Among NBD derivatives, N1‐(7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl)propane‐1,3‐diamine (NBD‐NH2) is found to bind selectively to the thymine base opposite an AP site in a DNA duplex with a binding affinity of 1.52×106 M ?1. From molecular modeling studies, it is suggested that the NBD moiety binds to thymine at the AP site and a protonated amino group tethered to the NBD moiety interacts with the guanine base flanking the AP site. Green fluorescent NBD‐NH2 is successfully applied for simultaneous G>T genotyping of PCR amplification products in a single cuvette in combination with a blue fluorescent ligand, 2‐amino‐6,7‐dimethyl‐4‐hydroxypteridine (diMe‐pteridine). |
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| Keywords: | abasic site DNA recognition DNA fluorescence single nucleotide polymorphism |
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