多个序列突变的筛查-HRMA（四）
替代凝胶电泳
标准是使用琼脂糖凝胶电泳和EB着色。通过片段的长度、纯度和条带的荧光量确定片段。HRMA是一种很有优势的技术；通过熔解图、存在其它熔解图的纯度（没有额外的熔解峰）和产生的大量荧光信号（图6）。使用HRMA的优势很明显；不需要灌胶、使用危险化学药品（EB），熔解比电泳速度快，数据分析可以自动完成。此外，HRMA是一种非破坏性的方法，当HRMA没能得到明确的结果时，片段依然可以在凝胶上进行分析。
图6.用HRMA替代凝胶电泳。A：5个不同PCR片段的分析；因为片段间有清晰的熔解图差异，所有五个片段可以在一个分析完成。当熔解图部分重叠，可以做到每个片段分析。B:PCR上可以进行96个不同样品的分析。一个PCR失败的荧光，其它的可以通过荧光水平估计。纯化，包括引物二聚体的存在，可以被HRMA差异图的分析检测出（未显示）。C：HRMA后插入384板PCR后，进行第二轮选择。几个清晰的熔解组确定，表示包含克隆的组有相同的插入。确认所有组的存在，第一次分析后确认分析需要分离和软件必须重新分组。重复这一过程直到所有组得到确定。
在我们实验室，HRMA迅速取代了凝胶电泳用于分析PCR产物。加入颜料后进行PCR（每10 μL体系加1 μL
10ⅹLC-Green染料），样品在管内95℃
5min，降至室温，然后熔解。图5显示了分析5个不同片段，都得到了清晰的熔解图。在熔解峰低Tm值处可以辨认处引物二聚体。OUT等（提交手稿）使用HRMA代替凝胶电泳检测扩增，同时在MUTYH基因测序前确定长期的PCR指导。作者成功地检测了几乎所有的期望突变达0.5%（1/200同源染色体）
克隆鉴定
一些实验产生一系列克隆需要测序确定。这些包括体外突变实验、甲基化研究、CDNA克隆确定不同剪接的水平或等位基因表达和噬菌体筛选。HRMA提供了一种有吸引力的工具筛选克隆，检测那些具有相同插入和变异。减少了大量的测序工作。图6C显示了第二次噬菌体筛选的结果。几个克隆组的实验结果具有相同的熔解图，显示实验成功地选择了几个不同的噬菌体克隆。随后每组的克隆代表测序验证了HRMA的结果；组内序列差异和测序确定[Pepers等2009]。以前，克隆插入确定是使用酶切和凝胶电泳，灵敏度低且实验繁琐[Verheesen
等, 2006]
结论
HRMA的优势使它迅速吸引了一批新用户。HRMA操作简单、简单、灵活、成本低，非破坏性、灵敏度和特异性高的特点是它成为了筛查患者致病突变的一种新方法。综上所述，HRMA有多个吸引力的其他应用，使它具有多种分析核酸的功能。由于HRMA依然是一项新技术，期待它有更好的发展。Fluidigm公司推出了一个纳升qPCR系统[Spurgeon等,
2008],目前推出了一个96个样品分析的PCR实验(例如，同时进行9261个实验)；设想这样一个系统时，将有助于HRMA。
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