Abstract：
High-resolution melt (HRM) analysis is a closed-tube technique for detecting single nucleotide polymorphisms (SNPs). However, it has limited use in high-resolution melting devices, even those with high thermal accuracy (HTA). In addition to the cost of switching to these specialized devices, the presence of nearest neighbour neutral changes (class III, IV SNPs and small indels) made HRM-based assays a challenging task due to reduced sensitivity. This study aimed to design a common modified competitive amplification of differently melting amplicons (CADMA)-based assay to address these challenges by generating allele-specific qPCR products that are detectable on most qPCR platforms. For this study, SNPs were selected from all four classes of SNPs (class I: C/T or G/A mutation; class II: C/A or G/T mutation; class III: G/C mutation; class IV: A/T mutation). A single base pair and 19 bp indels were also chosen to simulate how CADMA primers could be designed for indels of varying lengths. The melting temperatures (Tm) were determined using IDT oligoAnalyzer. qPCR and melt data acquisition were performed on the CFX96 qPCR platform, and the melt curve data were analyzed using Precision Melt software (Bio-Rad, USA). The clusters for different genotypes were successfully identified with the aid of the control samples, and Tm predictions were carried out using the uMelt batch and Tm online tools for comparison. Using HRM-qPCR assays based on the modified CADMA method, genotyping of various SNPs was successfully carried out. For some SNPs, similarly shaped melt curves were observed for homozygotes and heterozygotes, making shape-based genotype prediction difficult. The Tm values calculated via the Blake and Delcourts (1998) method were the closest to the experimental Tm values after adjusting for the salt concentration. Since HRM assays usually depend on the ΔTm caused by mutations, they are prone to a high error rate due to nearest neighbour neutral changes. The technique developed in this study significantly reduces the failure rates in HRM-based genotyping and could be applied to any SNP or indel in any platform. It is crucial to have a deep understanding of the melt instrument, its accuracy and the nature of the target (SNP class or indel length and GC content of the flanking region). Furthermore, the availability of controls is essential for a high success rate.
摘要：
高分辨率解链（HRM）分析是一种用于检测单核苷酸多态性（SNP）的闭管技术。然而,它在高分辨率熔化设备中的应用有限，即使是那些具有高热精度（HTA）。除了切换到这些专用设备的成本外，最近邻中性变化的存在(III类，IVSNP和小indel）由于灵敏度降低，使基于HRM的测定成为一项具有挑战性的任务。本研究旨在设计基于不同解链扩增子(CADMA)的测定的常见修饰竞争性扩增,以通过产生在大多数qPCR平台上可检测的等位基因特异性qPCR产物来解决这些挑战。对于这项研究，SNP选自所有四类SNP(I类:C/T或G/A突变;II类:C/A或G/T突变;III类:G/C突变;IV类:A/T突变)。还选择了单个碱基对和19bp的插入缺失来模拟如何为不同长度的插入缺失设计CADMA引物。使用IDT寡核苷酸分析仪测定熔融温度(Tm)。qPCR和解链数据采集在CFX96qPCR平台上进行，并使用PrecisionMelt软件(Bio-Rad，美国）。在对照样品的帮助下，成功地鉴定了不同基因型的簇，和Tm预测使用uMelt批次和Tm在线工具进行比较。使用基于改进的CADMA方法的HRM-qPCR检测，成功地进行了各种SNP的基因分型。对于某些SNP，观察到纯合子和杂合子的相似形状的解链曲线，使得基于形状的基因型预测变得困难。通过Blake和Delcourts(1998)方法计算的Tm值在调整盐浓度后最接近实验Tm值。由于HRM测定通常取决于突变引起的ΔTm，由于最近邻中性变化，它们容易出现高错误率。本研究中开发的技术显着降低了基于HRM的基因分型的失败率，并且可以应用于任何平台中的任何SNP或indel。对熔体仪器有深入的了解至关重要，其准确性和靶标的性质(侧翼区的SNP类或indel长度和GC含量)。此外，控制的可用性对于高成功率至关重要。
