改良碘化钾法提取冻存外周血基因组 DNA 的方法探讨

摘要

Objective To explore the modified potassium iodide method (Improved method) for extracting the long-term cryopreservation of peripheral blood genomic DNA. Methods The potassium iodide (KI) method was improved by increasing the blood volume from 100μL to 200μL, replacing the sterile distilled water with 0.9% NH4CL solution for destruction the red blood cells, increasing the 5 mol/L KI solution dosage(70μL) for cracking the white cells membrane, and centrifuging at low temperature(4℃ ). The modified potassium iodide method was used for extracting the genomic DNA from 82 blood samples reserved in -80℃, and the concentration and purity of the extracted DNA was compared with the KI method. Meanwhile the extracted DNA was used as the template to amplify CYP2C19 gene by PCR. Results The extracted DNA concentration and A260/A280 of the KI method and the improved method were 128.2±34.9μg/mL, 1.74±0.08 and 220±91.29μg/mL, 1.87±0.12, respectively. The DNA purity of improved method was higher, and there were significant statistically different (P<0.01). The amount of DNA extracted by the modified method was more than that of KI method. The genomic DNA extracted by improved method were amplified using PCR, which result were stable. Conclusion The genomic DNA extracted from the long-term cryopreservation of peripheral blood using improved KI method had higher quality, which were able to meet the demand on the clinical study of cryopreserved samples.%　　目的探讨用改良碘化钾法(改良法)提取长期冻存外周血基因组 DNA 的方法.方法对碘化钾(KI)法加以改良,包括：血量由100μL 增加到200μL；使用0.9% NH4CL 溶液代替无菌重蒸馏水裂解红细胞；增加裂解白细胞膜的5 mol/L KI 溶液的用量(为70μL)；低温(4℃)离心.然后用改良碘化钾法从82份-80℃冻存外周血标本中提取基因组 DNA.同时使用碘化钾法提取其中的30份血标本,比对两种方法提取 DNA 的浓度和纯度,并进行CYP2C19相关基因 PCR 扩增.结果两种方法所提取的 DNA 浓度和纯度分别是碘化钾法为128.2±34.9μg/mL 和 A260/A2801.74±0.08,改良法为220±91.29μg/mL 和 A260/A2801.87±0.12.改良法获得的 DNA 纯度更高,差异有统计学意义(P<0.01),通过增加样本量及对方法进行改良获得的 DNA 量较多.对改良后方法提取的基因组 DNA 进行 PCR 扩增,结果稳定.结论改良碘化钾法从长期冻存外周血中所提取的基因组 DNA 质量较高,能够满足对临床冻存样本研究的需求.