毛细管动态涂渍区带电泳模式对DNA分子定量方法的建立及应用  

Establishment and Application of A Quantitative Method for DNA with Dynamic Coating Capillary Zone Electrophoresis

作　　者：伊茂礼[1] 臧琴波[1] 沈佐君[2] 何晓东[2] 

机构地区：[1]青岛大学医学院附属烟台毓璜顶医院检验科,山东烟台264000 [2]安徽省临床检验中心,合肥230001

出　　处：《国际检验医学杂志》2010年第9期932-934,共3页International Journal of Laboratory Medicine

摘　　要：目的 在毛细管电泳仪上建立一种定量DNA的方法.方法 在聚乙烯吡咯烷酮（PVP）的＂动态＂涂渍作用前提下,用pUC19DNA/Mspl（HpaⅡ）为DNA标准品,荧光染料SYBR Green Ⅰ为DNA嵌合染料,建立了一种定量DNA的方法.结果 在0.5～500 ng/mL范围内方法具有良好的线性,相关系数r2为0.991 8,按信噪比S/N=3,本方法最低检测限为0.5 ng/mL.利用本方法好的线性、灵敏度和重现性,我们成功定量了外周血中提取的游离RNA的逆转录产物,试验结果显示,肺癌患者血循环中提取的RNA的逆转录产物的量明显高于健康者.结论 本方法不需要价格昂贵且寿命短的涂层柱,只需将含PVP的缓冲液充柱,再用不含PVP的缓冲液冲出即可,非常有潜力在临床和生物化学领域得到广泛应用.Objective To establish a method for quantification of DNA by capillary zone electrophoresis. Methods pUC19DNA/Mspl（Hpa Ⅱ ）Maker fragments were utilized for calibration and SYBR Green I as DNA intercalating dye CZE LIF was applied for the separation and quantification of DNA. Results Good linearity（R2=0. 9918）in the range of DNA concentration（0.5 41 000 ng/mL）and a detection limit of 0.5ng/ml for DNA（S/N=3）were obtained. Our data demonstrated that this approach had a good linearity with excellent sensitivity and satisfactory reproducibility in the quantification of DNA. This method was successful ly used to quantify the circulating cell free RNA for cancers. We observed that the cDNA levels of circulating cell free RNA quantity of cancers were significantly higher than that of healthy individuals. Conclusion Compared to previous methods, our protocol does not need expensive coated capillary, only needs to fill capillary with PVP solution. It would have a great potential application for both clinical and biochemical analysis.

关 键 词：电泳 毛细管 DNA 实验室技术和方法 

分 类 号：R440[医药卫生—诊断学]