| Co-Investigator(Kenkyū-buntansha) |
KAWABATA Shinnichirou Shimadzu Corporation Analytical Instruments, Produst Manager, 分析機器事業部, プロダクトマネージャー
TAMIYA Gen Tokai University School of Medicine, Assistant Researcher, 医学部, 助手 (10317745)
SHIINA Takashi Tokai University School of Medicine, Assistant Researcher, 医学部, 助手 (00317744)
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| Research Abstract |
When DNA molecules are subjected to MALDI-TOF-based mass spectrometry, signals obtained are weak with low sensitivity and resolution, especially for high molecular weight DNAs. Due to these reasons, DNAs of longer than 100 bp are difficult to be measured for detection of 2 bp differences. In order to overcome these problems and thereby to develop genotyping of microsatellite repeat polymorphisms by MALDI-TOF, we optimized various experimental conditions to make 2 bp differences distinguished precisely. At first, detailed conditions were investigated for purification of DNAs, DNA concentration and strength of X ray laser. As a result, resolution index of 101 (100 bp and 102 bp length could be distinguished) could be attained for synthesized poly dT with 100 bp. Further, studies on purification steps allowed to detect DNA with the length of up to 276 bp (PCR product). Also the conditions to enable genotyping of tri- to penta- nucleotide repeat microsatellites with high molecular weight by MALDI-TOF were identified. A new MALDI-TOF machine by the use of infrared rays was developed as an aim to apply much higher molecular weight DNAs. In addition, DNA chips with a small amount of mutiple samples spotted were devised and developed using cheap glass plate for high-throughput analysis. This mass spectrometry chips were found to be successfully applied to not only genotyping of microsatellite repeat polymorphisms but also of SNP (single nucleotide polymorphisms). Finally, IR laser-based MALDI-TOF mass spectrometry machine, which was expected to be suitable for longer DNA samples, was developed and confirmed for its effective measurement.
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