Dystrophin基因缺失检测微阵列构建及应用中技术优化的研究

目的建立Dystrophin基因缺失检测的DNA微阵列技术，并优化其构建及应用中的关键技术条件。方法提取样品基因组DNA，Klenow法随机引物扩增同时FITC荧光标记，并比较生物素标记法。将FITC荧光标记的核酸点样于不同方法活化处理的玻片上并用不同浓度的盐溶液洗脱，测试对DNA固定效率的影响。以分子克隆法获得的Dystrophin基因18个易缺失外显子cDNA片段为探针、APES和poly-Lys联合处理的玻片为基片，制备简易微阵列。分别与DMD/BMD患者及健康人荧光标记的基因组DNA杂交，检测微阵列的质量和评估结果的可靠性。结果APES和poly-Lys联合处理的玻片对核酸固定效率最高，核酸在洗涤过程中的脱落程度随洗液盐浓度的增加而增大；FITC标记步骤简便，生物素标记相对烦琐，但生物素标记杂交后的荧光强度明显高于FITC标记；微阵列杂交信号信噪比较好，各种对照结果满意，检测结果与PCR一致。结论优化了DNA微阵列构建及应用过程中基片表面活化、探针固定、靶基因DNA扩增标记、杂交条件、杂交信号检测分析等方法，为更好地应用微阵列进行Dystrophin基因诊断奠定基础。

Study on technical optimization of DNA microarray construction and application for Dystrophin gene analysis

Objective： Develop DNA microarray technique for Dystrophin gene deletion analysis, and optimize technique of microarray construction and application. Methods： Genomic DNA were prepared from blood samples, then amplified by random primer and labeled with FITC. To compare that labeled with Biotin. Glass slides were treated with APES and poly - Lysine respective or together, and the absorbent DNA sample labeled with FITC on them was washed by the solutions with different concentration of saline to examine the stability of absorbent DNA. 18 major deletion - prone exons of Dystrophin gene obtained by cloning were used as probes and were spotted on the slides treated with APES and poly - Lysine together. Such microarrays were hybridized with the FITC labeled genomic DNA of DMD/BMD patients or healthy people. Results were used to evaluate the mieroarray. Results： DNA absorbed on slides treated with APES and poly - Lysine together had the best absorption. During washing, the higher the saline concentration of the solution was, the more the absorbed DNA would be eroded. The hybridizing signals were prefect, and the hybridization results were coincidental with those of PCR. Conclusion： Activation on slide＇s surface, probe immobilization, amplification and labeling of target DNA, hybridization condition, and signal detection were optimized. Establish the foundation for applying DNA microarray to Dystrophin gene analysis.