Application of “Homogeneous Assay for Fluorescence Concentrated on Membrane” to the Analysis of the Substrate Specificity of Protease

Tsuchida, Shirou; Sügimura, Takashi; Yahara, Taemi; Nunome, Kana; Hamaue, Naoya; Aoki, Takashi

doi:10.1271/bbb.90822

Cited by 2 publications

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“…Alternative methods include capillary electrophoresis and melting curve analysis, both of which require expensive equipment [6][7][8][9][10][11]. To overcome these limitations, the "homogeneous assay for fluorescence concentrated on membrane" (HAFCOM), affinity analysis method we developed is applicable to designing a simple and novel genotyping method [12][13][14]. During the study, it was noticed that for hybridization forming a cytosine-guanine (CG) base pair between a probe possessing a fluorescein isothiocyanate (FITC) labeled terminal cytosine (C) and target DNA with a terminal guanine (G), FITC fluorescence intensity was dramatically reduced.…”

Section: Introductionmentioning

confidence: 99%

Photoinduced electron transfer detection method for identifying UGT1A1*28 microsatellites

et al. 2023

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During development of a novel detection method for the UDP-glucuronosyl transferase 1A1 (UGT1A1)*28, the fluorescence intensity of a dye conjugated to cytosine (C) at the end of a DNA strand decreased upon hybridization with guanine (G). This phenomenon is referred to as photoinduced electron transfer (PeT). Using this phenomenon, we devised a method for the naked-eye detection of UGT1A1*28 (thymine-adenine (TA)-repeat polymorphism). Fluorescently labeled single-stranded DNA (ssDNA) oligonucleotides (probes) were designed and hybridized with complementary strand DNAs (target DNAs). Base pair formation at the blunt end between fluorescently labeled C (probe side) and G (target side), induced dramatic fluorescence quenching. Additionally, when the labeled-CG pair formed near the TA-repeat sequence, different TA-repeat numbers were discriminated. However, obtaining enough target DNA for this probe by typical polymerase chain reaction (PCR) was difficult. To enable the practical use of the probe, producing sufficient target DNA remains problematic.

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