Li M, Palais R A, Zhou L, Wittwer C T
Department of Pathology, University of Utah School of Medicine, 383 Colorow Drive, Salt Lake City, UT 84108, USA.
Department of Pathology, University of Utah School of Medicine, 383 Colorow Drive, Salt Lake City, UT 84108, USA; Department of Mathematics, Utah Valley University, 800 W University Parkway, Orem, UT 84058, USA.
Anal Biochem. 2017 Dec 15;539:90-95. doi: 10.1016/j.ab.2017.10.015. Epub 2017 Oct 21.
High resolution DNA melting of PCR products is a simple technique for sequence variant detection and analysis. However, sensitivity and specificity vary and depend on many factors that continue to be defined. We introduce the area between normalized melting curves as a metric to quantify genotype discrimination. The effects of amplicon size (51-547 bp), melting rate (0.01-0.64 °C/s) and analysis method (curve shape by overlay vs absolute temperature differences) were qualitatively and quantitatively analyzed. To limit experimental variance, we studied a single nucleotide variant with identical predicted wild type and homozygous variant stabilities by nearest neighbor thermodynamic theory. Heterozygotes were easier to detect in smaller amplicons, at faster melting rates, and after curve overlay (superimposition), with some p-values <10. As heterozygote melting rates increase, the relative magnitude of heteroduplex contributions to melting curves increases, apparently the result of non-equilibrium processes. In contrast to heterozygotes, the interplay between curve overlay, PCR product size, and analysis method is complicated for homozygote genotype discrimination and is difficult to predict. Similar to temperature cycling in PCR, if the temperature control and temperature homogeneity of the solution are adequate, faster rates improve melting analysis, just like faster rates improve PCR.
聚合酶链反应（PCR）产物的高分辨率DNA熔解分析是一种用于序列变异检测与分析的简单技术。然而，其灵敏度和特异性各不相同，且取决于许多仍有待明确的因素。我们引入归一化熔解曲线之间的面积作为一种量化基因型区分的指标。对扩增子大小（51 - 547碱基对）、熔解速率（0.01 - 0.64℃/秒）和分析方法（通过叠加曲线形状与绝对温度差异）的影响进行了定性和定量分析。为限制实验方差，我们通过最近邻热力学理论研究了具有相同预测野生型和纯合变异稳定性的单核苷酸变异。在较小的扩增子中、较快的熔解速率下以及曲线叠加（重叠）后，杂合子更容易检测到，一些P值<10。随着杂合子熔解速率的增加，异源双链体对熔解曲线贡献的相对大小增加，这显然是非平衡过程的结果。与杂合子不同，对于纯合子基因型区分，曲线叠加、PCR产物大小和分析方法之间的相互作用较为复杂且难以预测。与PCR中的温度循环类似，如果溶液的温度控制和温度均匀性足够，更快的速率会改善熔解分析，就像更快的速率会改善PCR一样。
