| 本文已被：浏览 1772次 下载 1845次 |
码上扫一扫！ |
| 三种DNA抽提方法对新鲜血细胞及冻血细胞DNA抽提效能的比较 |
| 金晶1△,朱玲2△,高军1*,吴洪玉1,满晓华1,朱艳平1,龚燕芳1,李兆申1* |
|
|
(1. 第二军医大学长海医院消化内科, 上海 200433;
2. 上海体育学院中国乒乓球学院, 上海 200438
△共同第一作者
*通信作者) |
|
| 摘要: |
| 目的 比较3种血细胞DNA提取方法（进口试剂盒、国产试剂盒和改良苯酚抽提方法）对新鲜血细胞及冻血细胞DNA的提取效率及差异。 方法 收集20例胰腺癌患者5 mL外周血标本，经低速离心获得血细胞沉淀后分装，根据保存方式分为新鲜血细胞和冻血细胞。新鲜血细胞不经冻存，在12 h内迅速抽提DNA；冻血细胞在-40℃保存72 h后，室温解冻后抽提DNA。抽提方法分别采用进口试剂盒（Qiagen公司）、国产试剂盒（天根生化科技有限公司）和改良苯酚方法。琼脂糖电泳检测DNA片段的完整性，核酸蛋白检测仪测定DNA纯度和浓度，计算不同抽提方法的DNA得率。 结果 在获得的DNA完整性方面，改良苯酚方法抽提的基因组DNA断裂和降解较少，而进口和国产试剂盒抽提的DNA均有一定程度的降解小片段。在获得DNA的纯度方面，部分冻存血细胞无论应用进口试剂盒、国产试剂盒和改良苯酚方法，均表现出一定的污染杂峰。在DNA得率方面，无论新鲜血细胞和冻存血细胞，均是进口试剂盒得率最高，其次为国产试剂盒；冻血细胞的改良苯酚法DNA得率与国产试剂盒相当，新鲜血细胞的改良苯酚法DNA得率最低。在耗时和成本分析中，试剂盒较快但成本较高。 结论 改良苯酚抽提法可获得较进口和国产试剂盒片段更长、更完整的基因组DNA，虽然在得率和耗时方面稍逊色，但其成本较低，可推荐用于大规模、分批次、低成本冻存血细胞的基因组DNA抽提。 |
| 关键词: DNA抽提 外周血 血细胞 DNA质量 |
| DOI：10.3724/SP.J.1008.2014.00101 |
| 投稿时间：2013-06-16修订日期：2013-09-10 |
| 基金项目: |
|
| Three methods for extracting human genomic DNA from fresh and frozen blood samples: a comparison study |
| JIN Jing1△, ZHU Ling2△, GAO Jun1, WU Hong-yu1, MAN Xiao-hua1, ZHU Yan-ping1, GONG Yan-fang1, LI Zhao-shen1* |
(1. Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China;
2. The China Table Tennis College, Shanghai University of Sport, Shanghai 200438, China
△Co-first authors.
*Corresponding author.) |
| Abstract: |
| Objective To compare the efficacies of three methods (Qiagen kit, Tiangen kit and modified manual extraction) for extracting human genomic DNA from fresh and frozen blood samples. Methods Twenty samples of intravenous whole blood (5 mL/sample) were collected from pancreatic cancer patients and centrifuged. Each sample was then divided into fresh and frozen samples. Human genomic DNA was extracted from the fresh blood samples within 12 h by the three methods: Qiagen kit, Tiangen kit and modified manual extraction. The frozen sample was thawed to room temperature after preserved at -40℃ for 72 h and the DNA was extracted by the same three methods. The integrity of the DNA products was examined by agarose gel electrophoresis; the purity and concentration were examined by nucleic acid protein detector; and the cost and time spent of the 3 methods were evaluated. Results The DNA integrity by the modified manual method was better than those of the other two methods using kits. As for purity, some frozen samples presented ghost peaks of contamination. As for the yield of the DNA products, the Qiagen kit was superior to Tiangen kit for both fresh and frozen samples. The yield of modified manual method was similar to that of Tiangen kit for frozen samples, and was the lowest one for fresh samples. Methods using kit took less time but cost more. Conclusion The modified manual extraction can obtain longer and more intact genomic DNA compared with the other two methods. Although the modified manual extraction is inferior in yield and time saving, it costs less and is more suitable for genomic DNA extraction from large frozen samples. |
| Key words: DNA extraction blood blood cells DNA quantity |
