摘要
目的:运用原子力显微镜(atomic force microscope,AFM)表征K562/A02细胞多药耐药基因Mdr1启动子区域的DNA。材料与方法:PCR扩增K562/A02细胞Mdr1基因启动子区域769bp的DNA,产物纯化后,分别按终浓度为5ng/μl、10ng/μl、20ng/μl固定在用1ng/μlL型多聚赖氨酸预先处理过的新剥离的云母片制备成观测样本。原子力显微镜观测样本,获取扫描图像后追踪DNA分子轮廓,进行模数分析。结果:用原子力显微镜能够成功地表征K562/A02mdr1基因启动子区域769bp的DNA片段,获得清晰的二维及三维图像。我们推测原子力显微镜观测该长度的DNA样本的最佳浓度为10ng/ul左右,在此浓度下对其三维图像进行图像处理后分析的DNA分子轮廓结果如下:DNA分子链的长度为(260.13±2.29)nm(n=50),平均宽度为(11.88±0.92)nm,双链的平均高度为1.2nm。结论:原子力显微镜能够成功地表征K562/A02细胞Mdr1基因启动子区域的DNA,获得清晰的二维及三维图象,并且能够直观地分析DNA轮廓,为进一步在分子层面上探讨研究Mdr1基因遗传信息提供了一种新的简单、直观、可靠的方法。
Objective： To identificate 5＇ regulatory region DNA ofmdrl gene in K562/A02 cells by Atomic Force Microscopy （AFM）. Materials and Methods： Cultivate and PCR technique was used to amplify K562/A02 cells with the 769bp 5＇regulatory region DNA ofmdrl gene, which fragment from -755 to ＋14. The amplified products were purified by the PCR product purification kit, According to final concentration of 5-20 ng/μl, DNA samples were placed on a freshly cleaved mica which was treated by 1ng/μl L-polylysine. AFM was used＇to image and analysis target DNA fragment. Results： We estimated the optimum DNA concentration to yield well-absorbed DNA molecular on the mica surface was 10 ng/μl. The length of the DNA fragment measured by AFM image was （260.13 ±2.29） nm, the width was （11.88±0.92） nm （mean_±SD, n=50）, the mean height was 1.2nm. Conclusions： we report a new method of identifying 5＇ regulatory region DNA ofmdrl gene from image analysis using AFM. Based on the demonstrated capabilities of AFM, it is likely to become a powerful tool for gene expression, physical mapping, and conformation investigation.
出处
《中国医学物理学杂志》
CSCD
2006年第1期31-34,共4页
Chinese Journal of Medical Physics
基金
东南大学基金资助项目(XJ0490169)
