Background: Myotonic dystrophy type 1 (DM1) is caused by CTG repeat expansions in the DMPK gene and is the most common form of muscular dystrophy. Patients can have long delays from onset to diagnosis, since clinical signs and symptoms are often non-specific and overlapping with other disorders. Clinical genetic testing by Southern blot or triplet-primed PCR (TP-PCR) is technically challenging and cost prohibitive for population surveys. Methods: Here, we present a high throughput, low-cost screening tool for CTG repeat expansions using TP-PCR followed by high resolution melt curve analysis with saturating concentrations of SYBR GreenER dye. Results: We determined that multimodal melt profiles from the TP-PCR assay are a proxy for amplicon length stoichiometry. In a screen of 10,097 newborn blood spots, melt profile analysis accurately reflected the tri-modal distribution of common alleles from 5 to 35 CTG repeats, and identified the premutation and full expansion alleles. Conclusion: We demonstrate that robust detection of expanded CTG repeats in a single tube can be achieved from samples derived from specimens with minimal template DNA such as dried blood spots (DBS). This technique is readily adaptable to large-scale testing programs such as population studies and newborn screening programs.

中文翻译：

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使用融解曲线分析高通量筛选1型强直性营养不良中扩展的CTG重复序列

背景：1型强直性营养不良（DM1）是由DMPK基因中的CTG重复扩增引起的，是肌肉营养不良的最常见形式。由于临床体征和症状通常是非特异性的，并且与其他疾病重叠，因此患者从发病到诊断可能会有很长的延迟。通过Southern印迹或三联体引物PCR（TP-PCR）进行的临床基因检测在技术上具有挑战性，并且成本高昂，无法进行人群调查。方法：在这里，我们介绍了一种高通量，低成本的筛选工具，用于使用TP-PCR进行CTG重复扩增，然后使用饱和浓度的SYBR GreenER染料进行高分辨率熔解曲线分析。结果：我们确定从TP-PCR分析中得到的多峰熔体图谱是扩增子长度化学计量的替代指标。在10097个新生儿血斑的屏幕中，熔体谱分析准确地反映了5至35个CTG重复序列中常见等位基因的三峰分布，并鉴定了突变前和完全扩增等位基因。结论：我们证明，可以从衍生自具有最少模板DNA（例如干血斑（DBS））的标本的样品中，对单个试管中的CTG重复序列进行强健的检测。这种技术很容易适应大规模的测试程序，例如人口研究和新生儿筛查程序。