Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.
Curr Protoc. 2022 May;2(5):e427. doi: 10.1002/cpz1.427.
Fragile X syndrome and other fragile X-associated disorders are caused by the full-mutation (>200 copies) and premutation (55 to 200 copies) expansion, respectively, of the CGG short tandem repeat in the fragile X messenger ribonucleoprotein 1 (FMR1) gene. Clinical diagnostic laboratories use Southern blot analysis and polymerase chain reaction (PCR)-based tests to detect and/or size the FMR1 CGG repeats. The development of sensitive and high-throughput triplet-primed PCR (TP-PCR) assays has diminished the need to subject all samples to Southern blot analysis, which is both labor- and time-intensive. In this article, we describe two direct TP-PCR (dTP-PCR) assays for the detection of FMR1 CGG repeat expansions. We outline a protocol that is based on melting curve analysis of dTP-PCR amplicons for a rapid and cost-effective first-tier screening and identification of individuals with premutation and full-mutation expansions. We also describe a protocol that employs capillary electrophoresis to resolve the dTP-PCR amplicon fragments and to estimate the repeat sizes of normal (5 to 44 copies), intermediate (45 to 54 copies), and premutation alleles, as well as to detect full mutations and determine the structure of the FMR1 alleles. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Direct triplet-primed PCR master mix preparation and amplification of the FMR1 CGG repeat locus for melting curve analysis Basic Protocol 2: Melting curve analysis of direct triplet-primed PCR amplicons on the Rotor-Gene Q MD × 5plex high-resolution melt platform Alternate Protocol: Melting curve analysis of direct triplet-primed PCR amplicons on the LightCycler 480 system Basic Protocol 3: Generation of direct triplet-primed PCR melting curve analysis profiles Basic Protocol 4: Direct triplet-primed PCR master mix preparation and amplification of the FMR1 CGG repeat locus for capillary electrophoresis Basic Protocol 5: Generation of control FMR1 plasmids for direct triplet-primed PCR melting curve analysis Basic Protocol 6: Sanger sequencing assay to verify FMR1 CGG repeat size and structure of plasmid DNA controls.
脆性 X 综合征和其他脆性 X 相关疾病分别由脆性 X 信使核糖核蛋白 1 (FMR1) 基因中的 CGG 短串联重复序列的完全突变（>200 个拷贝）和前突变（55 至 200 个拷贝）扩展引起。临床诊断实验室使用 Southern 印迹分析和聚合酶链反应 (PCR) 为基础的测试来检测和/或确定 FMR1 CGG 重复序列的大小。敏感和高通量三引物 PCR (TP-PCR) 检测方法的发展减少了对所有样本进行 Southern 印迹分析的需要，Southern 印迹分析既费时又费力。在本文中，我们描述了两种用于检测 FMR1 CGG 重复扩展的直接 TP-PCR (dTP-PCR) 检测方法。我们概述了一种基于 dTP-PCR 扩增物熔解曲线分析的方案，用于快速、经济高效地筛选和鉴定前突变和完全突变的个体。我们还描述了一种使用毛细管电泳来解析 dTP-PCR 扩增片段并估计正常（5 至 44 个拷贝）、中间（45 至 54 个拷贝）和前突变等位基因重复大小以及检测完全突变和确定 FMR1 等位基因结构的方案。
