High throughput screening for expanded CTG repeats in myotonic dystrophy type 1 using melt curve analysis.
High throughput screening for expanded CTG repeats in myotonic dystrophy type 1 using melt curve analysis.
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DOI：
10.1002/mgg3.1619
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发表时间：
2021-04
影响因子：
2
通讯作者：
Weiss RB
中科院分区：
文献类型：
--
作者：
Butterfield RJ;Imburgia C;Mayne K;Newcomb T;Dunn DM;Duval B;Feldkamp ML;Johnson NE;Weiss RB
肌强直性营养不良1型（DM1）是由DMPK基因中CTG重复扩增引起的，是肌营养不良最常见的形式。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有很长时间的延迟。通过Southern blot或三重引物PCR （TP - PCR）进行临床基因检测在技术上具有挑战性，并且在人口调查中成本过高。在这里，我们提出了一种高通量，低成本的筛选工具，使用TP - PCR进行CTG重复扩增，然后使用饱和浓度的SYBR绿色染料进行高分辨率熔融曲线分析。我们确定TP‐PCR测定的多模态熔体剖面是扩增子长度化学计量学的代表。在10097个新生儿血斑中，熔体谱分析准确地反映了5 ~ 35个CTG重复序列的共同等位基因的三模态分布，并鉴定出了突变前和完全扩增等位基因。我们证明，扩增的CTG重复序列在单管中可以从具有最小模板DNA的标本（如干血斑（DBS））中获得的样品中实现稳健检测。这项技术很容易适用于大规模的测试项目，如人口研究和新生儿筛查项目。1型肌强直性营养不良是最常见的肌肉营养不良之一，由DMPK基因中CTG重复扩增引起。在这里，我们提出了一种高通量、低成本的筛选工具，使用TP - PCR检测CTG重复序列的扩增，然后使用饱和浓度的SYBR绿色染料进行熔融曲线分析。我们证明，扩增的CTG重复序列在单管中可以从具有最小模板DNA（如干血斑）的样本中获得准确而稳健的检测。
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 nonspecific 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. 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. 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. 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. Myotonic dystrophy type 1 is one of the most common forms of muscular dystrophy, caused by a CTG repeat expansion in the DMPK gene. Here, we present a high throughput, low‐cost screening tool to detect an expansion of the CTG repeat using TP‐PCR followed by melt curve analysis with saturating concentrations of SYBR GreenER dye. We demonstrate that accurate and 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.
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DOI：
10.1093/brain/awp236
发表时间：
2009-11
期刊：
Brain : a journal of neurology
影响因子：
--
作者：
Norwood FL;Harling C;Chinnery PF;Eagle M;Bushby K;Straub V
通讯作者：
Straub V
影响因子：
9.8
作者：
Tishkoff, SA;Goldman, A;Kidd, KK
通讯作者：
Kidd, KK
影响因子：
4.1
作者：
Lian, Mulias;Rajan-Babu, Indhu-Shree;Chong, Samuel S.
通讯作者：
Chong, Samuel S.
影响因子：
2.1
作者：
Do, ThuyQuynh N.;Street, Natalie;Bolen, Julie
通讯作者：
Bolen, Julie
影响因子：
5.8
作者：
Dwight, Zachary;Palais, Robert;Wittwer, Carl T.
通讯作者：
Wittwer, Carl T.