超声波破碎-高效液相色谱法定量检测核酸  

Application of High Performance Liquid Chromatography with Ultrasonic Splitting for DNA Quantification

作　　者：董莲华[1] 盛灵慧[1] 王晶[1] 黎朋[1] 

机构地区：[1]中国计量科学研究院,北京100013

出　　处：《分析化学》2011年第9期1442-1446,共5页Chinese Journal of Analytical Chemistry

基　　金：科技支撑项目(No.2008BAK41B01);博士科研专项基金(No.ATQDB0908)资助

摘　　要：采用超声波破碎结合高效液相色谱技术,建立了定量检测质粒DNA的方法,测量结果可以溯源至核苷酸标准物质。采用超声波破碎(功率300 W,频率24 kHz)技术将质粒DNA破碎成200～500 bp的小片段DNA,再用蛇毒磷酸二酯酶将其水解为4种核苷酸(dCMP:3.2 min;dTMP:4.7 min;dGMP:5.3 min,dAMP:6.8 min),将产物通过HPLC分离后,采用外标法进行定量分析。应用此方法对待测质粒pNK603进行定量分析,测定的4种核苷酸的摩尔百分比(A∶T=0.95,C∶G=0.98)与理论值接近。本方法的测定结果(47.24μg/g)与实时荧光定量聚合酶链式反应(PCR,45.98μg/g)和PicoGreen荧光染料法(46.02μg/g)的测定结果没有显著差异,表明建立的超声波-HPLC方法可以应用于质粒DNA的定量分析。A new traceable DNA quantification method by HPLC with ultrasonic splitting was set up in this study and the measurement can be traceable to nucleotides.First,the large fragment of DNA such as plasmid DNA,was cut into 200-500 bp by ultrasonic（power at 300 W,frequency at 24 kHz） and then the products were hydrolyzed to four kinds of nucleotide（dCMP： 3.2 min;dTMP： 4.7 min;dGMP： 5.3 min;dAMP： 6.8 min） by snake venom phosphodiesterase.Finally,the hydrolyzed products were separated and quantified by HPLC.The mole percentage of each nucleotide in plasmid pNK603 measured by HPLC（A∶T=0.95,C∶G=0.98） was close to its corresponding theoretical value.Additionally,there was no significant difference among the results determined by ultrasonic-HPLC（47.24 μg/g）,real time PCR（45.98 μg/g） and PicoGreen kit（46.02 μg/g）.This indicates the technique of ultrasonic-HPLC can be successfully applied to plasmid DNA quantification.

关 键 词：质粒 超声波破碎 高效液相色谱 荧光 聚合酶链式反应 

分 类 号：O629.7[理学—有机化学] O657.72[理学—化学]