Objective:To investigate the effects of different plasma separation strategies and DNA extraction methods on plasma DNA concentration in order to obtain the most suitable plasma DNA separation and purification strategy.Methods:The peripheral blood samples of 20 healthy people were collected for comparison of plasma separation with DNA extraction method.The plasma DNA concentration obtained from different centrifugation speed of plasma separation and three commonly used DNA extraction methods was measured by real-time quantitative PCR.The effect of different extraction methods on the recovery of different length of DNA fragments was also studied and compared.The differences in different groups were analyzed by student's t-test.The most suitable plasma DNA separation strategy was selected to detect and analyze fetal DNA in 10 abnormal pregnancy samples and 12 healthy pregnancy samples.Results:The results showed that the centrifugation speed had significant effect on the plasma separation and DNA extraction.The difference of plasma DNA concentration obtained with the three DNA extraction methods was statistically significant(P＜0.05).The plasma DNA concentration obtained by the three-step low speed method,the two-step low speed method and the one-step high speed method was(17.9±2.72)ng·mL-1,(30.4±4.26)ng·mL-1L and(38.4±11.87)ng·mL-1 respectively.Sample freezing had no significant effect on plasma separation results except for high-speed centrifugation.The results of three DNA extraction methods showed that the plasma DNA concentration obtained by kit method(20.02±1.26)ng·mL-1 was significantly lower than that obtained by phenol chloroform method(29.35±2.91)ng·mL-1 and sodium iodide method(24.2±1.08)ng·mL-1(P＜0.05).Except for the phenol-chloroform method,the other two extraction methods also had significant differences in the DNA fragments recovery(P＜0.05),and sodium iodide method was more suitable for the extraction of short DNA fragments.The detection results of plasma DNA from pregnant women with abnormal pregnancy showed that the concentration of SRY gene in the plasma of preeclampsia was significantly increased(P＜0.05).Conclusion:The two-step low speed centrifugation method for plasma separation and sodium iodide method for DNA extraction can be widely used for plasma DNA separation,purification,detection and analysis.Fetal DNA concentration in the plasma of preeclampsia pregnant women increases significantly,which is expected to be used for abnormal pregnancy screening.
plasma DNAextraction methodcentrifugal speedDNA fragment
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