Drexel University, School of Biomedical Engineering, Science, and Health Systems, Philadelphia, Pennsylvania, USA.
Analyst. 2013 Oct 21;138(20):6117-26. doi: 10.1039/c3an00384a.
Detection of mutated (MT) deoxyribonucleic acid (DNA) amongst the wild type (WT) requires the probe DNA (pDNA) that is complementary to the MT to discriminate the WT by one or two nucleotide mismatches. Traditionally this is achieved by raising the temperature to above the melting temperature (Tm) of the WT (TWT) but below that of the MT (TMT). However, a raised temperature is also accompanied by a weakened binding of the MT to the pDNA which can reduce the detection sensitivity. In this study, we investigated flow as a way to enhance MT detection specificity at a lower temperature. Gold-coated glass (GCG) slides immobilized with pDNA complementary to the target MT were placed at the center of the flow cell. The detection was done by flowing MT or WT at various concentrations followed by flowing 10(5) ml(-1) fluorescent reporter microspheres (FRMs) that were 6 μm in size and coated with reporter DNA complementary to the MT or WT but different from the pDNA at various flow rates and temperatures. The detection of MT or WT was characterized by counting the FRMs captured on the GCG. Hepatitis B virus 1762/1764 double mutation (HBV DM) was the model MT and the TMT and TWT were 47 °C and 22 °C, respectively. It was shown that at room temperature, flow initially increased the binding of both the MT and WT at lower flow rates but decreased the binding at flow rates ≥4 ml min(-1) due to the increase in the flow-induced impingement force on the FRMs to overcome the binding of the MT and the WT to the GCG at higher flow rates. At ≥30 °C the decrease in binding of the WT with an increasing flow rate was more than that of the MT because 30 °C was above the TWT but still well below the TMT. As a result, the detection of MT at 30 °C with a flow rate of 4 ml min(-1) was more specific than at 35 °C without flow. These results indicate that flow can diminish WT binding at a lower temperature than without flow and allow MT detection to occur at a lower temperature with high specificity.
检测野生型（WT）中的突变（MT）脱氧核糖核酸（DNA）需要与 MT 互补的探针 DNA（pDNA），通过一个或两个核苷酸错配来区分 WT。传统上，这是通过将温度提高到 WT 的熔点（TWT）以上但低于 MT（TMT）来实现的。然而，升高的温度也伴随着 MT 与 pDNA 的结合减弱，这可能会降低检测灵敏度。在这项研究中，我们研究了流动作为一种在较低温度下增强 MT 检测特异性的方法。金涂玻璃（GCG）载玻片固定有与靶 MT 互补的 pDNA，放置在流动池的中心。通过以不同浓度流动 MT 或 WT，然后以不同流速和温度流动 10（5）ml（-1）大小为 6 μm 且涂覆有与 MT 或 WT 互补但与 pDNA 不同的报告 DNA 的荧光报告微球（FRM）来进行检测。通过计数 GCG 上捕获的 FRM 来表征 MT 或 WT 的检测。乙型肝炎病毒 1762/1764 双突变（HBV DM）是模型 MT，TMT 和 TWT 分别为 47°C 和 22°C。结果表明，在室温下，流动最初会在较低流速下增加 MT 和 WT 的结合，但在流速≥4 ml min（-1）时会降低结合，因为 FRM 上的流动诱导撞击力会增加，以克服在较高流速下 MT 和 WT 与 GCG 的结合。在≥30°C 时，随着流速的增加，WT 的结合减少量大于 MT，因为 30°C 高于 TWT 但仍远低于 TMT。因此，在 4 ml min（-1）的流速下在 30°C 下检测 MT 比在没有流动的情况下在 35°C 下更具特异性。这些结果表明，流动可以在低于没有流动的温度下减少 WT 结合，并允许在低温下以高特异性进行 MT 检测。
