Microarray-based AMASE as a novel approach for mutation detection
Microarray-based AMASE as a novel approach for mutation detection
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DOI：
10.1016/j.mrfmmm.2004.03.009
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发表时间：
2004-10-04
影响因子：
2.3
通讯作者：
Lundeberg, J
中科院分区：
文献类型：
--
作者：
Käller, M;Ahmadian, A;Lundeberg, J
p53肿瘤抑制基因的改变是许多人类癌症病例中的重要事件。我们开发了一种新型的基于微阵列的方法，用于p53基因的重新测序和突变检测。该方法有助于快速和简单地扫描靶基因序列，并可以扩展到包括其他候选癌症基因。该方法采用之前描述的三磷酸腺苷双磷酸酶介导的等位基因特异性延伸反应（AMASE）。为了对所选区域进行重新测序，将具有不同3 '末端的四种延伸寡核苷酸用于每个碱基位置，并将它们共价连接到载玻片的表面。然后将扩增的单链DNA模板与阵列杂交，然后在腺苷三磷酸双磷酸酶存在下用荧光标记的dNTPs原位延伸。所用的模型系统是基于对p53基因外显子5中15 bp片段的分析。将突变评分为等位基因分数，计算为（wt）/（wt + mut）信号。当腺苷三磷酸双磷酸酶包括在野生型模板的延伸反应中时，平均等位基因分数为0.96。当用相同的野生型模板排除腺苷三磷酸双磷酸酶时，获得显著较低的等位基因分数。使用两个60-mer合成寡核苷酸来建立AMASE突变的可检测量，并且可以在所有点之间进行明确区分。还分析了来自不同阶段的皮肤恶性肿瘤的几个样品。本研究的结果表明，利用AMASE技术可以高效、准确地对p53基因进行全序列测定。(C)2004 Elsevier B. V.保留所有权利。
Alterations in the p53 tumor suppressor gene are important events in many cases of human cancers. We have developed a novel microarray based approach for re-sequencing and mutation detection of the p53 gene. The method facilitates rapid and simple scanning of the target gene sequence and could be expanded to include other candidate cancer genes. The methodology employs the previously described apyrase-mediated allele-specific extension reaction (AMASE). In order to re-sequence the selected region, four extension oligonucleotides with different 3'-termini were used for each base position and they were covalently attached to the glass slide's surface. The amplified single-stranded DNA templates were then hybridized to the array followed by in situ extension with fluorescently labeled dNTPs in the presence of apyrase. The model system used was based on analysis of a 15 bp stretch in exon 5 of the p53 gene. Mutations were scored as allelic fractions calculated as (wt)/(wt + mut) signals. When apyrase was included in the extension reactions of wild type templates, the mean allelic fraction was 0.96. When apyrase was excluded with the same wild type templates, significantly lower allelic fractions were obtained. Two 60-mer synthetic oligonucleotides were used to establish the detectable amount of mutations with AMASE and a clear distinction between all the points could be made. Several samples from different stages of skin malignancies were also analyzed. The results from this study imply the possibility to efficiently and accurately re-sequence the entire p53 gene with AMASE technology. (C) 2004 Elsevier B.V. All rights reserved.