论文部分内容阅读
目的优化慢性粒细胞白血病(Chronic myelogenous leukemia,CML)ssDNA适配子的PCR扩增条件。方法以合成的核苷酸适配子为模板并设计引物,根据引物解链温度(Tm值)设置不同的退火温度(38.0、39.2、41.2、44.2、48.3、51.5、53.6、55.0℃)、循环次数(12、16、20、24、28、30、35、38、40)及非限制性与限制性引物比例(1∶1、30∶1、50∶1、70∶1、80∶1、100∶1),进行PCR扩增,优化CML ssDNA适配子对称PCR及间接不对称PCR扩增条件,并验证优化的PCR条件的重复性。结果对称PCR扩增的最佳退火温度为44.2℃,最佳循环次数为30次;间接不对称PCR的最佳循环次数为38次,非限制性引物与限制性引物浓度比例为50∶1时,可获得理想的ssDNA和较少的dsDNA;优化的PCR条件重复性较好。结论优化了CML ssDNA适配子的PCR扩增条件,为筛选到特异性的ssDNA适配子提供了参考,也为后续试验奠定了基础。
Objective To optimize the PCR amplification conditions for ssDNA aptamers of chronic myelogenous leukemia (CML). Methods Using the synthesized nucleotide aptamers as template and designing primers, different annealing temperatures (38.0, 39.2, 41.2, 44.2, 48.3, 51.5, 53.6, 55.0 ℃) were set according to the melting temperature of primer (12,16,20,24,28,30,35,38,40) and non-limiting and limiting primer ratios (1: 1, 30: 1, 50: 1, 100: 1). PCR amplification was performed to optimize the CML ssDNA aptamer symmetric PCR and indirect asymmetric PCR amplification conditions, and to verify the reproducibility of the optimized PCR conditions. Results The optimal annealing temperature for symmetric PCR amplification was 44.2 ℃, the optimal number of cycles was 30, the optimal number of cycles for indirect asymmetric PCR was 38, and the ratio of non-limiting primer to restriction primer was 50:1 , The ideal ssDNA and less dsDNA can be obtained. The optimized PCR conditions are reproducible. Conclusion The PCR amplification conditions of CML ssDNA aptamers were optimized, which provided a reference for screening specific ssDNA aptamers and laid the foundation for subsequent experiments.