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Development of DNA chips for genotyping of genetic polymorphisms using mass spectrometry

开发利用质谱法对遗传多态性进行基因分型的 DNA 芯片

基本信息

批准号：
12794019
负责人：
INOKO Hidetoshi
金额：
$ 18.94万
依托单位：
Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for University and Society Collaboration
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12794019/
关键词：
mass spectrometry MALDI-TOF gene polymorphism SNP HLA Maicrosatellite slideglass DNA tip

项目摘要

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.

当DNA分子经受基于MALDI-TOF的质谱分析时，获得的信号弱，灵敏度和分辨率低，特别是对于高分子量DNA。由于这些原因，难以测量长于100 bp的DNA以检测2 bp的差异。为了克服这些问题，从而发展微卫星重复多态性的MALDI-TOF基因分型，我们优化了各种实验条件，使2 bp的差异准确区分。首先，对DNA纯化、DNA浓度和X射线激光强度等条件进行了详细的研究。结果表明，合成的100 bp的poly dT的分辨指数可达101（100 bp和102 bp长度可区分）。此外，对纯化步骤的研究允许检测长度高达276 bp的DNA（PCR产物）。还确定了能够通过MALDI-TOF对具有高分子量的三至五核苷酸重复微卫星进行基因分型的条件。为了应用更高分子量的DNA，开发了一种利用红外线的新型MALDI-TOF仪器。此外，我们还设计并研制了一种使用廉价的玻璃板进行高通量分析的多样品点样的DNA芯片。该质谱芯片不仅可用于微卫星重复多态性的基因分型，而且可用于单核苷酸多态性（SNP）的基因分型。最后，研制了适用于较长DNA样品的基于红外激光的MALDI-TOF质谱仪，并验证了其有效性。