慢性粒细胞白血病ssDNA适配子PCR扩增条件的优化  

Optimization of conditions for amplification of chronic myeloid leukemia ssDNA aptamer by PCR

作　　者：习静[1] 郭鹏[1] 韩跃武[1] 刘文伟 张雪燕[1] 

机构地区：[1]兰州大学基础医学院生物化学与分子生物学研究所,甘肃兰州730000 [2]甘肃奇正藏药有限公司,甘肃兰州730000

出　　处：《中国生物制品学杂志》2013年第3期422-424,429,共4页Chinese Journal of Biologicals

基　　金：甘肃省自然科学基金(1107RJZA219)

摘　　要：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 condition for amplification of chronic myeloid leukemia （CML） ssDNA aptamer by PCR. Methods CML ssDNA was amplified by PCR using the synthetic DNA aptamer as a template. The annealing temperatures （38. 0, 39. 2, 41.2, 44. 2, 48. 3, 51.5, 53. 6 and 55.0℃）, cycle numbers （12, 16, 20, 24, 28, 30, 35, 38 and 40） and the ratios of non-restrictive to restrictive primer concentrations （1 ： 1, 30 ： 1, 50 ： 1, 70 ： 1, 80 ： 1 and 100 ： 1 ） were set up according to the melting temperature （Tm） of designed primers, based on which the conditions for symmetric and asymmetric PCR were optimized, and verified for reproducibility. Results The optimal annealing temperature for symmetric PCR was 44. 2 ℃, while the optimal cycle number was 30. However, the optimal cycle number for asymmetric PCR was 38. When the ratio of non-restrictive and restrictive primer concentrations was 50 ： 1, ideal ssDNA and a small quantity of dsDNA were obtained. The optimized condition for PCR showed high reproducibility. Conclusion The condition for amplification of CML ssDNA by PCR was optimized, which provided a reference for screening of specific ssDNA aptamer and laid a foundation of further study.

关 键 词：慢性粒细胞白血病 ssDNA适配子 聚合酶链反应 优化 

分 类 号：R733.72[医药卫生—肿瘤]