Determination of microsatellite repeats in the human thyroid peroxidase (TPOX) gene using an automated gene analysis system with nanoscale engineered biomagnetite

Takahito Nakagawa, Kohei Maruyama, Haruko Takeyama, Tadashi Matsunaga

    Research output: Contribution to journalArticle

    6 Citations (Scopus)

    Abstract

    The number of repeat in the microsatellite region (AATG)5-14 of the human thyroid peroxidase gene (TOPX) was determined using an automated DNA analysis system with nano-scale engineered biomagnetite. Thermal melting curve analysis of DNA duplexes on biomagnetite indicated that shorter repeat sequences (less than 9 repeats) were easily discriminated. However, it was difficult to determine the number of repeats at more than nine. In order to improve the selectivity of this method for the longer repeats, a "double probe hybridization assay" was performed in which an intermediate probe was used to replace a target repeat sequence having more than 9 repeats with a shorter sequence possessing less than 9 repeats. Thermal probe melting curve analyses and Tm determination confirmed that the target with 10 repeats was converted to 5 repeats, 11 repeats converted to 4 and 12 to 3, respectively. Furthermore, rapid determination of repeat numbers was possible by measuring fluorescence intensities obtained by probe dissociation at 56 and 66 °C, and 40, 60 and 80 °C for signal normalization.

    Original languageEnglish
    Pages (from-to)2276-2281
    Number of pages6
    JournalBiosensors and Bioelectronics
    Volume22
    Issue number9-10
    DOIs
    Publication statusPublished - 2007 Apr 15

    Fingerprint

    Iodide Peroxidase
    Microsatellite Repeats
    Freezing
    Hot Temperature
    Genes
    DNA
    Melting
    Fluorescence
    Assays

    Keywords

    • Automated system
    • Bacterial magnetic particles (BacMps)
    • DNA hybridization
    • Melting temperature
    • Microsatellite

    ASJC Scopus subject areas

    • Biotechnology
    • Analytical Chemistry
    • Electrochemistry

    Cite this

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    abstract = "The number of repeat in the microsatellite region (AATG)5-14 of the human thyroid peroxidase gene (TOPX) was determined using an automated DNA analysis system with nano-scale engineered biomagnetite. Thermal melting curve analysis of DNA duplexes on biomagnetite indicated that shorter repeat sequences (less than 9 repeats) were easily discriminated. However, it was difficult to determine the number of repeats at more than nine. In order to improve the selectivity of this method for the longer repeats, a {"}double probe hybridization assay{"} was performed in which an intermediate probe was used to replace a target repeat sequence having more than 9 repeats with a shorter sequence possessing less than 9 repeats. Thermal probe melting curve analyses and Tm determination confirmed that the target with 10 repeats was converted to 5 repeats, 11 repeats converted to 4 and 12 to 3, respectively. Furthermore, rapid determination of repeat numbers was possible by measuring fluorescence intensities obtained by probe dissociation at 56 and 66 °C, and 40, 60 and 80 °C for signal normalization.",
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    author = "Takahito Nakagawa and Kohei Maruyama and Haruko Takeyama and Tadashi Matsunaga",
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    AU - Nakagawa, Takahito

    AU - Maruyama, Kohei

    AU - Takeyama, Haruko

    AU - Matsunaga, Tadashi

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    N2 - The number of repeat in the microsatellite region (AATG)5-14 of the human thyroid peroxidase gene (TOPX) was determined using an automated DNA analysis system with nano-scale engineered biomagnetite. Thermal melting curve analysis of DNA duplexes on biomagnetite indicated that shorter repeat sequences (less than 9 repeats) were easily discriminated. However, it was difficult to determine the number of repeats at more than nine. In order to improve the selectivity of this method for the longer repeats, a "double probe hybridization assay" was performed in which an intermediate probe was used to replace a target repeat sequence having more than 9 repeats with a shorter sequence possessing less than 9 repeats. Thermal probe melting curve analyses and Tm determination confirmed that the target with 10 repeats was converted to 5 repeats, 11 repeats converted to 4 and 12 to 3, respectively. Furthermore, rapid determination of repeat numbers was possible by measuring fluorescence intensities obtained by probe dissociation at 56 and 66 °C, and 40, 60 and 80 °C for signal normalization.

    AB - The number of repeat in the microsatellite region (AATG)5-14 of the human thyroid peroxidase gene (TOPX) was determined using an automated DNA analysis system with nano-scale engineered biomagnetite. Thermal melting curve analysis of DNA duplexes on biomagnetite indicated that shorter repeat sequences (less than 9 repeats) were easily discriminated. However, it was difficult to determine the number of repeats at more than nine. In order to improve the selectivity of this method for the longer repeats, a "double probe hybridization assay" was performed in which an intermediate probe was used to replace a target repeat sequence having more than 9 repeats with a shorter sequence possessing less than 9 repeats. Thermal probe melting curve analyses and Tm determination confirmed that the target with 10 repeats was converted to 5 repeats, 11 repeats converted to 4 and 12 to 3, respectively. Furthermore, rapid determination of repeat numbers was possible by measuring fluorescence intensities obtained by probe dissociation at 56 and 66 °C, and 40, 60 and 80 °C for signal normalization.

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