常备试剂法提取人外周血DNA

A Common Reagents Protocol for DNA Extraction from Human Peripheral Blood Samples

目的探讨使用一种常备试剂提取外周血DNA,而无需PK、毒性有机溶剂或特殊缓冲液。方法分别使用常备试剂法、试剂型kit法和柱式型kit法提取外周血DNA,进而比较3种方法的DNA产量和质量。结果使用常备试剂法,提取200μL人外周血的DNA量为（5.77±3.49）μg,低于试剂型kit法（P〈0.05）,但与柱式型kit法（P=0.10）差异无统计学意义。常备试剂法提取的DNA样品,A230/A260为0.49±0.05,A260/A280为1.98±0.10,片段大于15 kb,Alu I、Bam HI-HF酶切完全,PCR扩增稳定、高效,DNA质量与两种kit法差异无统计学意义。常备试剂法的每样品耗费低,操作时间约30-40 min。结论常备试剂法能够保证外周血DNA产量和质量,试剂方便易得,操作快速、安全,且费用低廉,可作为一般实验室备选方案。

Objective To propose a common reagents protocol（ CRP） for DNA extraction from human peripheral blood samples. This CRP does not contain enzymes like proteinase-K,hazardous solvents like phenol,and special buffers like chaotropic agents. Methods Human peripheral blood samples were treated with the CRP,DNAzol BD Reagent Kit and Pure Link Genomic DNA Mini Kit,respectively. Then the yield and quality of DNA extracted were evaluated and compared. Results The average yield of DNA isolated by CRP was（ 5. 77 ± 3. 49） μg that was lower than cha Pure Link Genomic DNA Mini Kit（ P〈0. 05）,but the same as DNAzol BD Reagent Kit（ P = 0. 10）. The A230 / A260 was 0. 49 ± 0. 05,and A260 / A280 was 1. 98 ± 0. 10. Gel electrophoresis of the isolated DNA samples showed that the DNA fragments were above 15 kb. The isolated DNA was successfully digested by Bam HI-HF or Alu I restriction enzymes,and PCR amplification was also performed completely,indicating the DNA produced by CRP had acceptable quality. The CRP could be completed within 30 - 40 min. Furthermore,all the reagents were common and the most cost-effective of all methods. Conclusion The efficiency,speed,safety,and costeffectiveness make this CRP as an appropriate alternative to existing protocols for extracting DNA from peripheral blood in conventional laboratories.