Detection of single nucleotide polymorphisms by minisequencing on a polypyrrole DNA chip designed for medical diagnosis
Detection of single nucleotide polymorphisms by minisequencing on a polypyrrole DNA chip designed for medical diagnosis
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DOI：
10.1038/labinvest.3700387
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发表时间：
2006-03-01
影响因子：
5
通讯作者：
Lopez-Crapez, E
中科院分区：
文献类型：
--
作者：
Ho-Pun-Cheung, A;Choblet, S;Lopez-Crapez, E
随着遗传信息及其与人类疾病的关系的日益可用性，在医学诊断领域中对可以进行多个标记的平行基因分型的技术的需求日益增长。在本文中，我们报告了一种新的灵活的基于微阵列的方法，其目的是廉价，准确，并适应于常规分析的发展。MICAM（R）（医学分析微系统）DNA芯片的构建是基于在金电极上携带寡核苷酸探针的导电聚合物膜的受控电合成。首先，接枝探针的可接近的3 'OH-末端直接用于与荧光标记的链终止剂进行单模板依赖性核苷酸延伸反应。然后，记录对照和感兴趣的探针上掺入的双脱氧核苷酸的荧光以评估碱基识别。在这里，我们提出的方法来分配TP 53（肿瘤蛋白p53）密码子72多态性的基因型的发展及其应用分析的基因组DNA从细胞系和人类结直肠样本。通过对聚吡咯DNA芯片进行微测序获得的基因分型结果与通过聚合酶链反应-限制性片段长度多态性和直接测序获得的数据100%一致。该方法已成功应用于TP 53基因杂合性缺失的检测。
With the increasing availability of genetic information and its relationship to human diseases, there is a growing need in the medical diagnostic field for technologies that can proceed to the parallel genotyping of multiple markers. In this paper, we report the development of a new flexible microarray-based method that aims to be inexpensive, accurate, and adapted to routine analysis. The construction of the MICAM(R) (MICrosystem for Analysis in Medicine) DNA chip is based on the controlled electrosynthesis of a conducting polymer film bearing oligonucleotide probes on gold electrodes. First, accessible 3'OH-ends of grafted probes are directly used to conduct single template-dependent nucleotide extension reactions with fluorescence-labeled chain terminators. Then, the fluorescence of incorporated dideoxynucleotides on controls and probes of interest are recorded to assess base calling. Here, we present the development of the methodology to assign the genotype of TP53 (tumor protein p53) codon 72 polymorphism and its application to analysis of genomic DNA from cell lines and from human colorectal samples. The genotyping results obtained by minisequencing on the polypyrrole DNA chip were 100% concordant with data obtained by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing. Moreover, the developed probe array assay has been successfully applied to the detection of TP53 loss of heterozygosity.