Ericsson Olle, Sivertsson Asa, Lundeberg Joakim, Ahmadian Afshin
AlbaNova University Center, Department of Molecular Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden.
Electrophoresis. 2003 Oct;24(19-20):3330-8. doi: 10.1002/elps.200305583.
We have developed an array-based resequencing method to determine genetic alterations in putative cancer genes. The method relies on that the specificity of DNA polymerase in allele-specific extensions can be enhanced by terminating the extension reactions with apyrase and that a tiling set of primers are synthesized covering the investigated gene sequence. We report on such apyrase-mediated allele-specific primer extension (AMASE) assay as a method suitable for high-throughout resequencing and mutation detection in tumor suppressor genes and oncogenes. In the experimental setup, primers complementary to codons 12, 13 and codon 61 of the N-ras proto-oncogene were spotted onto glass slides. Overlapping sense and anti-sense primers were designed so that complementary primers for all possible mutations in each base position were investigated. The extension reactions were performed in a single step following hybridization of target DNA to the immobilized primers on the array surface. Mutation detection limits and the possibility of quantifying the mutations were investigated using synthetic oligonucleotides. In addition, 64 clinical samples were sequenced and 16 of these showed mutations in the N-ras gene.
我们开发了一种基于阵列的重测序方法，以确定假定癌症基因中的基因改变。该方法基于这样的原理：通过用硫酸腺苷酸双磷酸酶终止延伸反应，可以增强DNA聚合酶在等位基因特异性延伸中的特异性，并且合成了覆盖所研究基因序列的平铺引物集。我们报道了这种硫酸腺苷酸双磷酸酶介导的等位基因特异性引物延伸（AMASE）测定法，它是一种适用于肿瘤抑制基因和癌基因的高通量重测序和突变检测的方法。在实验设置中，将与N-ras原癌基因的第12、13密码子和第61密码子互补的引物点样到载玻片上。设计了重叠的正义和反义引物，以便研究每个碱基位置上所有可能突变的互补引物。在靶DNA与阵列表面固定的引物杂交后，一步进行延伸反应。使用合成寡核苷酸研究了突变检测限和定量突变的可能性。此外，对64份临床样本进行了测序，其中16份显示N-ras基因存在突变。
