Multiplex PCR liquid chromatography assay for detection of gene rearrangements:: application to RB1 gene -: art. no. e139
Multiplex PCR liquid chromatography assay for detection of gene rearrangements:: application to RB1 gene -: art. no. e139
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DOI：
10.1093/nar/gnh137
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Houdayer, C
中科院分区：
文献类型：
--
作者：
Dehainault, C;Laugé, A;Houdayer, C
大基因重排的筛选是分子医学的重要组成部分，但也具有挑战性。各种强大的方法可以检测全基因缺失，但将无法检测到更微妙的重排，可能涉及一个单一的外显子。在本文中，我们描述了一种新的，通用的和强大的方法来评估外显子拷贝数，称为多重PCR/液相色谱法（MP/LC）。使用未标记的引物扩增多个外显子，然后通过离子对反相高效液相色谱法（IP-RP-HPLC）分离，并使用柱后嵌入染料通过荧光检测进行定量。每个靶标的相对峰强度直接反映外显子拷贝数。这项新技术被用于筛选一组121名无关的视网膜母细胞瘤患者，这些患者先前使用参考策略进行了测试。MP/LC对所有缺失进行了正确评分，并证明了先前未检测到的RB 1重复，这是首次描述。MP/LC似乎是一种简单，通用和具有成本效益的方法，这是特别相关的变性HPLC（DHPLC）的用户，因为它拓宽了DHPLC系统的可用应用范围。
Screening for large gene rearrangements is established as an important part of molecular medicine but is also challenging. A variety of robust methods can detect whole-gene deletions, but will fail to detect more subtle rearrangements that may involve a single exon. In this paper, we describe a new, versatile and robust method to assess exon copy number, called multiplex PCR/liquid chromatography assay (MP/LC). Multiple exons are amplified using unlabeled primers, then separated by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and quantitated by fluorescent detection using a post-column intercalation dye. The relative peak intensities for each target directly reflect exon copy number. This novel technique was used to screen a panel of 121 unrelated retinoblastoma patients who were tested previously using a reference strategy. MP/LC correctly scored all deletions and demonstrated a previously undetected RB1 duplication, the first to be described. MP/LC appears to be an easy, versatile, and cost-effective method, which is particularly relevant to denaturing HPLC (DHPLC) users since it broadens the spectrum of available applications on a DHPLC system.