样品制备与扩增条件对寡核苷酸芯片杂交结果的影响  

Influence of Sample Preparation and Amplification Conditions on Oligonuleotide Chip

作　　者：陈慧[1] 高华方[2] 马雪梅[3] 杨渝珍[1] 

机构地区：[1]华中科技大学同济医学院基础医学院生物化学与分子生物学系,武汉430030 [2]清华大学生命科学院,北京100084 [3]生物芯片北京国家工程研究中心,北京100084

出　　处：《华中科技大学学报（医学版）》2004年第4期409-411,422,515,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：武汉市科委重大课题资助项目 (No .2 0 0 12 0 0 910 5 5 )

摘　　要：目的　以HLA A小阵列检测芯片为平台 ,确定HLA A寡核苷酸芯片检测中最优化实验条件。方法　观察了样品制备方法 (传统酚 氯仿法、白细胞裂解液法、Fe2 O3 纳米磁珠 )、不对称PCR制备ssDNA的参数、PCR产物纯化方法对杂交结果的影响。结果　 3种不同的基因组提取方法均获得良好的特异性PCR扩增结果 ,纳米磁珠应用于样品提取和PCR扩增获得成功 ,实验条件需进一步完善 ;在不对称PCR最佳实验条件为反向引物与正向引物浓度比 2 5∶1,正向引物浓度为 0 0 4 μmol/L ,PCR灵敏度可达ng (纳克 )模板DNA ,Qiagen纯化试剂盒效果略强于其它两种 ,但差异无显著性。结论　本研究初步探讨HLA A生物芯片样品制备条件 ,并取得理想结果 ,为寡核苷酸生物芯片的实际操作提供一定的参考和指导。Objective To study the influence of sample preparation on HLA-A oligonuleotide chip to optimize the experimental conditions. Methods Different ways of sample preparation were observed based on HLA-A biochip, such as two different genome DNA extraction methods (Phenol-chloroform method and leucocyte lysis method), nano-magnetic beads (Fe 2O 3) used in nucleus cell separation, the primers concentration and ratio in asymmetric PCR and three PCR product purification ways. Results By using the two different DNA extraction ways, satisfactory hybridization results were obtained. The nano-beads succeeded in sample extraction and PCR amplification, and the condition should be further improved. The proper primers concentration and ratio have been fixed to 25∶1 (reverse primer to forward primer, the concentration of forward primer was 0.04 μmol/L) to make better results. The hybridization result by Qiagen purification column was better than that of the other two, but the difference was not significant. Conclusion The sample preparation conditions by using HLA-A oligonucleotide biochip were studied and ideal results were obtained.

关 键 词：样品制备 寡核苷酸芯片 PCR产物 异丙醇 

分 类 号：R977.4[医药卫生—药品]