Arrayed primer extension: Solid-phase four-color DNA resequencing and mutation detection technology
Arrayed primer extension: Solid-phase four-color DNA resequencing and mutation detection technology
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DOI：
10.1089/109065700316408
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发表时间：
2000-03-01
期刊：
影响因子：
--
通讯作者：
Metspalu, A
中科院分区：
文献类型：
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作者：
Kurg, A;Tonisson, N;Metspalu, A
介绍了阵列引物延伸（APEX）技术和应用，描述了一个集DNA芯片和模板制备、阵列上多重引物延伸、荧光成像和数据分析于一体的集成系统。该方法基于通过 5' 端固定在玻璃表面上的寡核苷酸阵列。患者 DNA 通过 PCR 扩增、酶消化并与固定引物退火，从而使用四种独特的荧光标记双脱氧核苷酸促进模板依赖性 DNA 聚合酶延伸反应的位点。通过引物位点颜色代码的变化来检测突变。该技术应用于10种常见β-地中海贫血突变的分析。正确鉴定了 9 个患者 DNA 样本，每个样本都携带不同的突变，以及 4 个野生型 DNA 样本。该技术的信噪比平均为 40:1，能够以高置信度识别杂合突变。 APEX 方法可应用于任何 DNA 靶标，以有效分析突变和多态性。
The technology and application of arrayed primer extension (APEX) is presented, We describe an integrated system with DNA chip and template preparation, multiplex primer extension on the array, fluorescence imaging, and data analysis. The method is based upon an array of oligonucleotides, immobilized via the 5' end on a glass surface. A patient DNA is amplified by PCR, digested enzymatically, and annealed to the immobilized primers, which promote sites for template-dependent DNA polymerase extension reactions using four unique fluorescently labeled dideoxy nucleotides. A mutation is detected by a change in the color code of the primer sites. The technology was applied to the analysis of 10 common beta-thalassemia mutations. Nine patient DNA samples, each of which carries a different mutation, and four wild-type DNA samples were correctly identified. The signal-to-noise ratio of this technology is, on the average, 40:1, which enables the identification of heterozygous mutations with a high confidence level. The APEX method can be applied to any DNA target for efficient analysis of mutations and polymorphisms.