Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, California 95616, USA.
Genome Res. 2013 Jan;23(1):121-8. doi: 10.1101/gr.141705.112. Epub 2012 Oct 11.
The human fragile X mental retardation 1 (FMR1) gene contains a (CGG)(n) trinucleotide repeat in its 5' untranslated region (5'UTR). Expansions of this repeat result in a number of clinical disorders with distinct molecular pathologies, including fragile X syndrome (FXS; full mutation range, greater than 200 CGG repeats) and fragile X-associated tremor/ataxia syndrome (FXTAS; premutation range, 55-200 repeats). Study of these diseases has been limited by an inability to sequence expanded CGG repeats, particularly in the full mutation range, with existing DNA sequencing technologies. Single-molecule, real-time (SMRT) sequencing provides an approach to sequencing that is fundamentally different from other "next-generation" sequencing platforms, and is well suited for long, repetitive DNA sequences. We report the first sequence data for expanded CGG-repeat FMR1 alleles in the full mutation range that reveal the confounding effects of CGG-repeat tracts on both cloning and PCR. A unique feature of SMRT sequencing is its ability to yield real-time information on the rates of nucleoside addition by the tethered DNA polymerase; for the CGG-repeat alleles, we find a strand-specific effect of CGG-repeat DNA on the interpulse distance. This kinetic signature reveals a novel aspect of the repeat element; namely, that the particular G bias within the CGG/CCG-repeat element influences polymerase activity in a manner that extends beyond simple nearest-neighbor effects. These observations provide a baseline for future kinetic studies of repeat elements, as well as for studies of epigenetic and other chemical modifications thereof.
人类脆性 X 智力低下 1 号（FMR1）基因在其 5'非翻译区（5'UTR）中含有一个（CGG）（n）三核苷酸重复序列。该重复序列的扩展导致许多具有不同分子病理学的临床疾病，包括脆性 X 综合征（FXS；完全突变范围，大于 200 CGG 重复）和脆性 X 相关震颤/共济失调综合征（FXTAS；前突变范围，55-200 重复）。这些疾病的研究受到现有 DNA 测序技术无法对扩展的 CGG 重复序列进行测序的限制，特别是在完全突变范围内。单分子实时（SMRT）测序提供了一种与其他“下一代”测序平台在根本上不同的测序方法，非常适合长重复 DNA 序列。我们报告了第一个在完全突变范围内扩展 CGG 重复 FMR1 等位基因的序列数据，这些数据揭示了 CGG 重复片段对克隆和 PCR 的干扰作用。SMRT 测序的一个独特特征是其能够实时提供连接的 DNA 聚合酶核苷添加率的信息；对于 CGG 重复等位基因，我们发现 CGG-DNA 对脉冲间隔具有链特异性影响。这种动力学特征揭示了重复元件的一个新方面；即，CGG/CCG-重复元件中的特定 G 偏好在某种程度上影响聚合酶活性，这种影响超出了简单的最近邻效应。这些观察结果为重复元件的未来动力学研究以及对其表观遗传和其他化学修饰的研究提供了基线。
