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目的:建立一种经济、快速且高质量提取人体外周凝血DNA的方法。方法:摸索最佳的匀浆条件,对外周凝血块进行匀浆,采用KI法对匀浆液进行基因组DNA的提取,通过凝胶电泳、单重PCR和多重PCR检测凝血基因组DNA的提取产量和质量,并分别与常规的凝血基因组DNA提取方法,即蛋白酶K消化法,以及提取抗凝血基因组DNA的KI法进行比较分析。结果:最佳的匀浆条件为:39000 rmp,15秒。在此条件下提取的基因组DNA完整性好,纯度和产量与蛋白酶K消化法提取凝血DNA和KI法提取抗凝血DNA的结果相比,没有统计学差异。单重PCR和多重PCR也获得了理想的扩增结果。结论:与常规的外周凝血提取方法相比(蛋白酶K消化法),本方法节省了时间和成本,能快速、经济、有效地提取外周凝血基因组DNA,可用于后续的科研和临床诊断需要,解决了部分科研机构血液基因组DNA的样本来源问题。
OBJECTIVE: To establish an economical, rapid and high quality method for extracting DNA from human peripheral blood. Methods: The optimal conditions of homogenization were explored, and the clot was homogenized. The KI method was used to extract genomic DNA from the homogenate. The yield and quality of clotting genomic DNA were determined by gel electrophoresis, single-PCR and multiplex PCR , Respectively, and compared with the conventional clot genomic DNA extraction method, namely, the digestion method of proteinase K, and the KI method for extracting anticoagulant genomic DNA. Results: The best homogenization conditions were 39000 rmp for 15 seconds. The genomic DNA extracted under these conditions had good integrity, purity and yield compared with the results obtained by digestion of coagulation DNA by proteinase K digestion and DNA extraction by antimicrobial DNA by KI, and there was no statistical difference. Single PCR and multiplex PCR also obtained the ideal amplification results. CONCLUSION: Compared with routine peripheral blood coagulation extraction method (proteinase K digestion method), this method saves time and cost, can rapidly and economically and effectively extract peripheral blood clotting genomic DNA, and can be used for subsequent research and clinical diagnosis. Part of the research organization of blood genomic DNA sample source.