三种常用抗凝剂处理血液样本后DNA提取方法探讨

Discussion on the Extraction Method of DNA from Blood Samples Treated with Three Common Anticoagulants

目的:探究血液样本在经过三种常见抗凝剂以不同浓度处理后,适宜的DNA提取方法。方法:使用肝素钠、柠檬酸钠、EDTA-Na2三种抗凝剂以1 mg/ml,2 mg/ml,4 mg/ml,8 mg/ml四种浓度剂量对血液样本进行抗凝处理,分别采用直接扩增法、Chelex-100法和硅珠法三种不同的DNA提取方法提取血液样本中的DNA,随后进行荧光复合扩增及STR自动分型,使用直接计数法对STR分型结果进行评价,并进行统计学分析和结果比较。结果:采用三种提取方法均能提取3种抗凝剂处理的血样中的DNA,并得到不同效果的STR分型。直接扩增法处理抗凝剂浓度为1 mg/ml,2 mg/ml,4 mg/ml的血样时,STR分型结果无显著性差异(P > 0.05),抗凝剂浓度为8 mg/ml时,结果有显著性差异(P 0.05)。结论:在研究的范围内,Chelex-100法和硅珠法提取的血液样本DNA质量不受三种抗凝剂的影响。血液样本经高浓度(≥8 mg/ml)抗凝剂处理后的DNA提取方法,不宜选用直接扩增法。

Objective: To explore the appropriate method of DNA extraction from blood samples treated with different concentrations of three common anticoagulants. Methods: Heparin sodium, sodium citrate, and EDTA-Na2 were used to anticoagulant the blood samples at four concentrations of 1 mg/ml, 2 mg/ml, 4 mg/ml, and 8 mg/ml. DNA was extracted from blood samples by three different DNA extraction methods, including direct amplification method, Chelex-100 method and silicon bead method, respectively. Then fluorescence multiplex amplification and automatic STR typing were performed. STR typing results were evaluated by direct counting method, and the results were statistically analyzed and compared. Results: The three extraction methods can be employed for isolating DNA from blood samples treated with different anticoagulants, resulting in the acquisition of distinct STR profiles. No significant differences were observed in STR typing results using the direct amplification method when the anticoagulant concentrations were 1 mg/ml, 2 mg/ml, and 4 mg/ml (P > 0.05). However, a significant difference was observed at an anticoagulant concentration of 8 mg/ml (P 0.05). Conclusion: Within the scope of the study, the DNA quality of blood samples extracted by Chelex-100 method and silica bead method was not affected by the three anticoagulants. Direct amplification is not suitable for DNA extraction from blood samples treated with high concentration of anticoagulant (≥8 mg/ml).