Improved fluorescent PCR-based assay for sizing CGG repeats at the FRAXA locus
Improved fluorescent PCR-based assay for sizing CGG repeats at the FRAXA locus
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DOI：
10.1515/cclm.1999.065
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发表时间：
1999-04-01
影响因子：
6.8
通讯作者：
Feldmann, D
中科院分区：
文献类型：
--
作者：
Houdayer, C;Lemonnier, A;Feldmann, D
脆性X综合征是最常见的涉及智力低下的遗传性疾病，通常由FMR1基因第一外显子中扩大的CGG重复引起。因此，在精神发育迟滞人群中寻找FRAXA基因的CGG扩展已成为神经儿科实践中的常规调查。因此，我们开发了一种基于荧光PCR的重复序列测定方法，作为一种费时费力的Southern杂交的替代方法。该方法使用反向荧光标记的引物，以及添加了二甲基亚砜和7-去氮杂-dGTP的扩展长模板聚合酶链式反应系统(TM)(罗氏)。它可以精确地确定男性和女性的CGG重复数，从正常到突变前的大小范围内的等位基因，并检测男性的全部突变。我们相信，这种允许高样本吞吐量的PCR方法，对于智力低下男性的一线筛查是有用的，可能还会辅以Southern杂交分析，以评估大突变等位基因的甲基化状态。
Fragile X syndrome is the most frequent heritable genetic disease involving mental retardation and is usually caused by an expanded CGG repeat in the first exon of the FMR1 gene. Therefore, searching for CGG expansion at the FRAXA locus among the mentally retarded has become a routine investigation in neuropaediatric practice. Consequently, we have developed a fluorescent PCR-based assay for sizing repeats as an alternative to laborious and time-consuming Southern blot. The procedure utilises a reverse fluorescent labelled primer, and the Expand Long Template PCR system(TM) (Roche) with addition of dimethylsulfoxide and 7-deaza-dGTP. It allows precise determination of the CGG repeat number in males and females for alleles from normal to premutation size range and detection of full mutations in males. We believe that this PCR protocol, allowing a high sample throughput, is useful for first-line screening among mentally retarded males, possibly complemented by Southern blot analysis to assess the methylation status of large mutated alleles.