Improved PCR based methods for detecting C9orf72 hexanucleotide repeat expansions.
Improved PCR based methods for detecting C9orf72 hexanucleotide repeat expansions.
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DOI：
10.1016/j.mcp.2016.06.001
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发表时间：
2016-08
影响因子：
3.3
通讯作者：
Warner JP
中科院分区：
文献类型：
--
作者：
Cleary EM;Pal S;Azam T;Moore DJ;Swingler R;Gorrie G;Stephenson L;Colville S;Chandran S;Porteous M;Warner JP
由于C9 orf 72六核苷酸重复扩增的富含GC的重复性质，基于PCR的检测方法具有挑战性。已经报道了PCR的几个局限性，克服这些局限性有助于确定致病范围。还需要开发改进的重复引物PCR测定，其允许即使在重复区域周围存在基因组变异的情况下也进行检测。我们已经优化了C9 orf 72六核苷酸重复扩增的PCR条件，使用甜菜碱作为共溶剂和特定的循环条件，包括缓慢的斜坡和高变性温度。我们已经开发了一种侧翼测定法，以及用于重复扩增的3′和5′端的重复引物PCR测定法，当它们一起使用时，即使在重复的3′端存在基因组变异的情况下，也提供了一种用于检测是否存在大于1000个重复的扩增的稳健策略。使用我们的检测，我们已经检测到47/442苏格兰ALS患者的重复扩增。此外，我们建议在临床诊断环境中联合使用这些检测。已经进行了跨C9 orf 72重复扩增高达900个重复的侧翼PCR。针对C9 orf 72重复扩增的3′和5′端描述的RP-PCR测定。通过RP-PCR对镶嵌样品的扩增进行稳健检测。RP-PCR的局限性。
Due to the GC-rich, repetitive nature of C9orf72 hexanucleotide repeat expansions, PCR based detection methods are challenging. Several limitations of PCR have been reported and overcoming these could help to define the pathogenic range. There is also a need to develop improved repeat-primed PCR assays which allow detection even in the presence of genomic variation around the repeat region. We have optimised PCR conditions for the C9orf72 hexanucleotide repeat expansion, using betaine as a co-solvent and specific cycling conditions, including slow ramping and a high denaturation temperature. We have developed a flanking assay, and repeat-primed PCR assays for both 3′ and 5′ ends of the repeat expansion, which when used together provide a robust strategy for detecting the presence or absence of expansions greater than ∼100 repeats, even in the presence of genomic variability at the 3′ end of the repeat. Using our assays, we have detected repeat expansions in 47/442 Scottish ALS patients. Furthermore, we recommend the combined use of these assays in a clinical diagnostic setting. Flanking PCR across C9orf72 repeat expansions up to 900 repeats has been performed. RP-PCR assays described for both 3′ and 5′ ends of the C9orf72 repeat expansion. Robust detection of expansions by RP-PCR for mosaic samples. Limitations of RP-PCR described.