Detection of epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) using a fully automated system with a nano-scale engineered biomagnetite
A fully automated system using nano-scale engineered biomagnetite was developed to detect mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC). Bacterial magnetic particles (BacMPs) were isolated from the magnetic bacterium Magnetospirillum magneticum AMB-1 and conjugated to streptavidin. Biotin-labeled target PCR products were then captured with the BacMPs, hybridized with the detection probe and detected by fluorescence signaling. The process was performed using a newly designed automated processor equipped with an XYZ mobile arm containing a 96-way automated pipetter, reagent dispenser and fluorescence detector. Two types of somatic mutations (in-frame deletions and point substitutions) in the EGFR gene were successfully identified within 3.5 h using this system, suggesting that this system could be used in clinical tests of EGFR gene mutations in lung cancer, and potentially other cancer, patients. Additionally, a very low mutation rate could be detected in these samples. (c) 2006 Elsevier B.V. All rights reserved.
Determination of microsatellite repeats in the human thyroid peroxidase (TPOX) gene using an automated gene analysis system with nanoscale engineered biomagnetite
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 T,, 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 degrees C, and 40, 60 and 80 degrees C for signal normalization. (c) 2006 Elsevier B.V. All rights reserved.
Single Nucleotide Polymorphism Detection in Aldehyde Dehydrogenase 2 (ALDH2) Gene Using Bacterial Magnetic Particles Based on Dissociation Curve Analysis.
K. Maruyama, H. Takeyama, E. Nemoto, T. Tanaka, K. Yoda, T. Matsunaga
Biotechnol Bioeng
87
687
-
694
2004.09
[Refereed]
Authorship:Lead author
DNA固定化磁性細菌粒子を用いた底面磁気分離型全自動SNP検出システムの構築
丸山浩平
博士論文(東京農工大学大学院工学研究科)
2004.03
Authorship:Lead author
Development and evaluation of an automated workstation for single nucleotide polymorphism discrimination using bacterial magnetic particles
T Tanaka, K Maruyama, K Yoda, E Nemoto, Y Udagawa, H Nakayama, H Takeyama, T Matsunaga
We designed an automated workstation for magnetic particle-based single nucleotide polymorphism (SNP) discrimination of ALDH genotypes. Bacterial magnetic particles (BMPs) extracted from Magnetospirillum magneticum AMB-1 were used as DNA carriers. The principle for SNP discrimination in this study was based on fluorescence resonance energy transfer (FRET) between FITC (donor) and POPO-3 (acceptor) bound to double-stranded DNA. The workstation is equipped with a 96-way automated pipetter which collects and dispenses fluids as it moves in x- and z-directions. The platform contains a disposable tip rack station, a reagent vessel serving as a stock for POPO-3 and FITC-labeled probes and a reaction station for a 96-well microtiter plate. BMPs were collected by attaching a neodymium iron boron sintered (Nd-Fe-B) magnet on the bottom of the microtiter plate. This system permits the simultaneous heating and magnetic separation of 96 samples per assay. The genotypes ALDH2*1 and ALDH2*2 were discriminated by calculating the relative fluorescence intensities on BMPs. (C) 2003 Elsevier B.V. All rights reserved.
Single-nucleotide polymorphism analysis using fluorescence resonance energy transfer between DNA-labeling fluorophore, fluorescein isothiocyanate, and DNA intercalator, POPO-3, on bacterial magnetic particles
H Nakayama, A Arakaki, K Maruyama, H Takeyama, T Matsunaga
To develop an analytical system for single-nucleotide polymorphisms (SNPs), the fluorescence resonance energy transfer (FRET) technique was employed on a bacterial magnetic particle (BMP) surface. A combination of fluorescein isothiocyanate (FITC; excitation 490 nm/emission 520 nm) labeled at the 5' end of DNA and an intercalating compound (POPO-3, excitation 534 nm/emission 570 nm) was used to avoid the interference from light scattering caused by nanoparticles. After hybridization between target DNA immobilized onto BMPs and FITC-labeled probes, fluorescence from POPO-3, which was excited by the energy from the FITC, was detected. The major homozygous (ALDH2*1), heterozygous (ALDH2*11*2), and minor homozygous (ALDH2*2) genotypes in the blood samples were discriminated by this method. The assay described herein allows for a simple and rapid SNP analysis using a fully automated system. (C) 2003 Wiley Periodicals, Inc.
Journal of Bioscience and Bioengineering
95
(
1
)
21
-
26
2003
[Refereed]
View Summary
Bacterial and artificial magnetic particles were modified using a polyamidoamine (PAMAM) dendrimer and outer shell amines determined. Bacterial magnetic particles were the most consistently modified. Transmission electron microscopic (TEM) analysis showed that the artificial magnetic particles were structurally damaged by the modification process including sonication. Furthermore, laser particle analysis of the magnetite also revealed damage. Small quantities of dendrimer-modified bacterial magnetic particles were used to extract DNA from blood. The efficiency of DNA recovery was consistently about 30 ng of DNA using 2-10 μg of dendrimer-modified bacterial magnetite. This technique was fully automated using newly developed liquid handling robots and bacterial magnetic particles.
Detection of epidermal growth factor receptor (EGFR) gene mutations in non-small cell lung cancer (NSCLC) with an automated system using bacterial magnetic particles
K. Maruyama, K. Ohshima, H. Takeyama, T. Mochizuki, T. Matsunaga
Biosensors 2006: The Ninth World Congress on Biosensors
(Toronto)
Presentation date:
2006.05
Microsatellite repeats detection using bacterial magnetic particles
T. Nakagawa, K. Maruyama, T. Tanaka, H. Takeyama, T. Matsunaga
Biosensors 2006: The Ninth World Congress on Biosensors
(Toronto)
Presentation date:
2006.05
ストレプトアビジン固定化バイオナノマグネタイトを用いた結核菌薬剤耐性遺伝子の検出
内田宣邦, 丸山浩平, 中川敬仁, 竹山春子, 川口竜二, 松永是
2006年電気化学会第73回大会
(首都大学東京)
Presentation date:
2006.04
磁性細菌粒子を用いたストレプトマイシン耐性結核菌の遺伝子変異検出とその自動化
丸山浩平, 内田宣邦, 田中剛, 竹山春子, 川口竜二, 松永是
第28回日本分子生物学会年会
(ヤフードーム(旧 福岡ドーム))
Presentation date:
2005.12
表面修飾磁性細菌粒子を用いた全自動DNA判別システムによるハイスループットSNP検出
丸山浩平, 田中剛, 竹山春子, 加藤貴彦, 松永是
2005年電気化学秋季大会
(千葉大学)
Presentation date:
2005.09
アミノシラン修飾磁性細菌粒子を用いたDNA抽出と自動SNP検出システムへの応用
橋本礼輔, 丸山浩平, 田中剛, 竹山春子, 松永是
2005年電気化学会第72回大会
(熊本大学)
Presentation date:
2005.04
磁性細菌粒子を用いた骨粗鬆症感受性遺伝子群の全自動SNP検出
丸山浩平, 橋本礼輔, 根本越男, 田中 剛, 依田 聖, 加藤貴彦, 竹山春子, 松永是
第8回日本化学会バイオテクノロジー部会
(甲南大学)
Presentation date:
2004.11
表面修飾磁性細菌粒子を用いた卓上型全自動DNA配列判別システムの開発と生物種判別への適用
丸山浩平, 根本越男, 田中剛, 依田聖, 竹山春子, 張成年, 松永是
日本生物工学会平成16年度大会
(名城大学)
Presentation date:
2004.09
バイオナノ磁性粒子を用いた底面分離型全自動SNP検出システムによる骨粗鬆症感受性遺伝子多型の検出
丸山浩平, 根本越男, 依田聖, 竹山春子, 松永是
日本化学会シンポジウム
(関西学院大学)
Presentation date:
2004.03
バイオナノ磁性粒子を用いた全血からのTGF-β1遺伝子SNP検出システムの開発
丸山浩平, 根本越男, 田中 剛, 依田 聖, 加藤貴彦, 竹山春子, 松永 是
日本化学会生体機能関連化学部会・バイオテクノロジー部会合同シンポジウム
(熊本大学)
Presentation date:
2003.10
High-Throughput SNP Discrimination in TGF-beta1 Gene Using a Fully Automated SNP Detection System with Nano-Biomagnetic Particles.
K. Maruyama, H. Takeyama, E. Nemoto, T. Tanaka, K. Yoda, T. Matsunaga
SBS 9th Annual Conference and Exhibition
(Portland)