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

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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

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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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title = "Determination of microsatellite repeats in the human thyroid peroxidase (TPOX) gene using an automated gene analysis system with nanoscale engineered biomagnetite",
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.",
keywords = "Automated system, Bacterial magnetic particles (BacMps), DNA hybridization, Melting temperature, Microsatellite",
author = "Takahito Nakagawa and Kohei Maruyama and Haruko Takeyama and Tadashi Matsunaga",
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T1 - Determination of microsatellite repeats in the human thyroid peroxidase (TPOX) gene using an automated gene analysis system with nanoscale engineered biomagnetite

AU - Nakagawa, Takahito

AU - Maruyama, Kohei

AU - Takeyama, Haruko

AU - Matsunaga, Tadashi

PY - 2007/4/15

Y1 - 2007/4/15

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.

KW - Automated system

KW - Bacterial magnetic particles (BacMps)

KW - DNA hybridization

KW - Melting temperature

KW - Microsatellite

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