DNA紫外吸收定量法中干扰校正用沉淀剂存在的问题及改进方法
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摘要
核酸定量分析的紫外吸收法中,采用钼酸铵-过氯酸沉淀大分子核酸,以校正酸溶性小分子物质的干扰。对钼酸铵-过氯酸沉淀剂存在的问题进行了分析探讨,并研究了以乙醇作为沉淀剂的改进方法,对乙醇的pH值、用量、沉淀时间、离心速度和离心时间等因素进行了考察,确定了乙醇作为沉淀剂的检测方法和条件。试验结果显示,钼酸铵-过氯酸沉淀剂在260 nm处有较强的光吸收,严重干扰测定结果,同时,结果重现性差。采用pH 3. 0的95%乙醇作为沉淀剂,校正小分子物质的紫外吸收干扰,测定结果相对误差可控制在5%以下,样品回收率达(97. 7±2. 1)%,方法稳定可靠,所用试剂简单、安全、操作简便,可用于DNA样品的常规定量检测及教学实验。
In UV absorption,it is commonly to use molybdate-perchloric acid precipitant to depositnucleic acid molecules in order to correct the interference caused by undistinguishable UV absorptionwith small molecules,it has been a routine experimental method for nucleic acid quantification andhas been recorded many times in teaching materials. In this paper,the problems of molybdate-perchloric acid as precipitant has been discussed and an improvement method which used ethanol asprecipitant was proposed. The detection method and conditions were determined through researchingon the pH value of ethanol,the amount of ethanol,the precipitation time,the centrifugal speed andthe centrifugal time. The results showed that the molybdate-perchloric acid precipitant had a strongabsorption at 260 nm and this might result in detection error and poor reproducibility. However,withpH 3. 0 and 95% ethanol as a substitute precipitant,it had smaller error(below 5%)and bettersample recovery(97. 7 ± 2. 1)%. The simplification,safety,easy operation and reliability featuresmake this a good method for conventional quantification of DNA samples and also a good experimentexample for teaching.
In UV absorption,it is commonly to use molybdate-perchloric acid precipitant to depositnucleic acid molecules in order to correct the interference caused by undistinguishable UV absorptionwith small molecules,it has been a routine experimental method for nucleic acid quantification andhas been recorded many times in teaching materials. In this paper,the problems of molybdate-perchloric acid as precipitant has been discussed and an improvement method which used ethanol asprecipitant was proposed. The detection method and conditions were determined through researchingon the pH value of ethanol,the amount of ethanol,the precipitation time,the centrifugal speed andthe centrifugal time. The results showed that the molybdate-perchloric acid precipitant had a strongabsorption at 260 nm and this might result in detection error and poor reproducibility. However,withpH 3. 0 and 95% ethanol as a substitute precipitant,it had smaller error(below 5%)and bettersample recovery(97. 7 ± 2. 1)%. The simplification,safety,easy operation and reliability featuresmake this a good method for conventional quantification of DNA samples and also a good experimentexample for teaching.
引文
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[2]芶琳,单志.生物化学实验[M]. 2版.成都:西南交通大学出版社,2015:8.
[3]周春丽,刘华,李玉萍. 2种提取猪肝基因组DNA方法的比较[J].安徽农业科学,2009,37(18):8355-8356.
[4] ALONSO A,MARTíN P,ALBARRáN C,et al. Real-time PCR designs to estimate nuclear and mitochondrial DNA copynumber in forensic and ancient DNA studies[J]. Forensic Sci Int,2004,139(2):141-149.
[5] WHITE R A,BLAINEY P C,FAN H C,et al. Digital PCR provides sensitive and absolute calibration for high through-putsequencing[J]. BMC Genomics,2009,10:116.
[6] HANDT G,MENSCHIKOWSKI M,LEHMANN W,et al. Digitale PCR in der labordiagnostik[J]. BIOspektrum,2015,21(5):507-510.