数字PCR技术对HEK293细胞系基因组DNA的定量  

Quantitative study of genomic DNA in HEK293 cell line by digital PCR

作　　者：毕华[1] 问芬芬 陶磊[1] 魏玲慧 杨靖清[1] 卢宁 秦玺[1] 梁成罡[1] BI Hua;WEN Fenfen;TAO Lei;WEI Linghui;YANG Jingqing;LU Ning;QIN Xi;LIANG Chenggang(National Institutes for Food and Drug Control,Beijing 100050,China;不详)

机构地区：[1]中国食品药品检定研究院,北京100050 [2]君研生物科技(山西)有限公司,山西太原030006

出　　处：《中国生物制品学杂志》2025年第2期190-196,203,共8页Chinese Journal of Biologicals

基　　金：山西省重点研发计划(202202130501006)。

摘　　要：目的通过微流体芯片式数字PCR技术对HEK293细胞基因组DNA进行定量研究,为更加准确地定量基因以及基因组DNA相关检测提供新思路。方法首先通过柱提法获得HEK293细胞DNA,经琼脂糖凝胶电泳进行鉴定及纯度分析,再采用传统分光光度法、Qubit法和微流体芯片式数字PCR技术分别对HEK293细胞基因组DNA进行定量并进行统计分析。结果分光光度法测定HEK293细胞基因组DNA浓度为100.08 ng/μL,Qubit法测定值为93.98 ng/μL,数字PCR法检测拷贝数为29722.81 copies/μL,按照1个人类单拷贝基因组约3.3 pg粗略回算,分光光度和Qubit法换算的拷贝数分别约为30327和28479 copies/μL,数字PCR法测定值与分光光度法测定值的偏差仅为2%,而与Qubit法测定值的偏差仅为4%。结论本研究通过数字PCR、分光光度和Qubit法对HEK293细胞基因组进行DNA测定并比较,为DNA的定量提供了一种新的测定方法及思路,同时也为其他核酸类物质的定量提供了参考。Objective To perform quantirative study of HEK293 cell genomic DNA by microfluidic chip-based digital PCR technology,and to provide new ideas for more accurate gene quantification and genomic DNA related detection.Methods Firstly,the HEK293 cell DNA was obtained by Spin Column,identified and analyzed for the purity by agarose gel electrophoresis.Then,the genomic DNA of HEK293 cells was quantified by traditional ultraviolet spectrophotometry,Qubit fluorescence quantification and microfluidic chip-based digital PCR technology and statistically analyzed.Results The concentration of genomic DNA in HEK293 cells was detected as 100.08 ng/μL by ultraviolet spectrophotometry,93.98 ng/μL by Qubit fluorescence quantification and the copy number was determined as 29722.81 copies/μL by digital PCR.Roughly calculated based on human single-copy genome of about 3.3 pg,the converted copy numbers of spectrophotometry and Qubit fluorescence quantification were about 30327 and 28479 copies/μL,respectively.The deviation of the digital PCR detection value from the spectrophotometry detection value was only 2%,and the deviation from the Qubit detection value was only4%.Conclusion In this study,HEK293 genomic DNA was detected and compared by digital PCR,spectrophotometry and Qubit fluorescence,which provides a new detection method and idea for DNA quantification,and also provides a certain reference for the quantification of other nucleic acids.

关 键 词：HEK293细胞 数字PCR 宿主DNA DNA定量 

分 类 号：R917[医药卫生—药物分析学]