Mutational scanning of large genes by extensive PCR multiplexing and two-dimensional electrophoresis: application to the RB1 gene.
Mutational scanning of large genes by extensive PCR multiplexing and two-dimensional electrophoresis: application to the RB1 gene.
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DOI：
10.1093/hmg/5.6.755
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发表时间：
1996-06
影响因子：
3.5
通讯作者：
N. V. van Orsouw;D. Li;P. van der Vlies;H. Scheffer;C. Eng;C. Buys;F. Li;J. Vijg
中科院分区：
文献类型：
--
作者：
N. V. van Orsouw;D. Li;P. van der Vlies;H. Scheffer;C. Eng;C. Buys;F. Li;J. Vijg
随着已鉴定的人类疾病基因数量的迅速增加，开发准确、经济高效的突变检测已成为当务之急。在这里，我们提出了广泛的PCR多路复用和二维（2-D） DNA电泳相结合，以筛选视网膜母细胞瘤（RB1）肿瘤抑制基因的26个外显子突变。在二维电泳中，片段在非变性梯度凝胶和变性梯度凝胶中分别按照大小和碱基对序列进行分离。所有的目标片段，设计为具有最佳的熔融特性，在两步PCR （6-plex长PCR预扩增和随后的25-plex短PCR）中制备，然后进行异源双工。然后在单一实验条件下对PCR扩增子的混合物进行二维电泳。通过这种设计，在33名双侧视网膜母细胞瘤患者中检测到18个不同外显子中35个先前鉴定的突变。这些结果表明，这种格式的二维电泳提供了一种普遍适用的、实用的、快速的、高精度的大基因诊断方法，可用于广谱的可能的致病突变。
With the rapid increase in the number of identified human disease genes, the development of accurate and cost-efficient mutation tests has become opportune. Here we present a combination of extensive PCR multiplexing and two-dimensional (2-D) DNA electrophoresis to screen for mutations in 26 exons of the retinoblastoma (RB1) tumor suppressor gene. In 2-D electrophoresis, fragments are separated according to size and base pair sequence in non-denaturing and denaturing gradient gels, respectively. All target fragments, designed to have optimal melting characteristics, were prepared in a two-step PCR (a 6-plex long-PCR pre-amplification and a subsequent 25-plex short-PCR) followed by heteroduplexing. The mixture of PCR amplicons was then subjected to 2-D electrophoresis under a single set of experimental conditions. With this design, 35 previously identified mutations in 18 different exons were detected in 33 bilateral retinoblastoma patients. These results suggest that 2-D electrophoresis in this format provides a generally applicable, practical and fast way to diagnose with high accuracy large genes for a broad spectrum of possible disease-causing mutations.