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

当DNA分子进行基于maldi - tof的质谱分析时，获得的信号较弱，灵敏度和分辨率较低，特别是对于高分子量的DNA。由于这些原因，长度超过100 bp的dna很难检测到2 bp的差异。为了克服这些问题，利用MALDI-TOF对微卫星重复多态性进行基因分型，我们对各种实验条件进行了优化，以精确区分2 bp的差异。首先，对DNA的纯化条件、DNA浓度和X射线激光强度进行了详细的研究。结果表明，对于长度为100 bp的合成聚dT，其分辨指数可达101（分别为100 bp和102 bp）。此外，纯化步骤的研究允许检测长度达276 bp的DNA （PCR产物）。并确定了利用MALDI-TOF进行高分子量三至五核苷酸重复微卫星基因分型的条件。开发了一种利用红外线的新型MALDI-TOF机器，目的是应用更高分子量的dna。此外，利用廉价的玻璃板设计和开发了少量多个样本斑点的DNA芯片，用于高通量分析。该质谱芯片不仅可以成功地用于微卫星重复多态性的基因分型，还可以用于单核苷酸多态性的基因分型。最后，研制了基于红外激光的MALDI-TOF质谱仪，该仪器有望用于更长的DNA样品，并证实了其有效的测量效果。

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.