Pastinen T, Kurg A, Metspalu A, Peltonen L, Syvänen A C
Department of Human Molecular Genetics, National Public Health Institute, Helsinki, Finland.
Genome Res. 1997 Jun;7(6):606-14. doi: 10.1101/gr.7.6.606.
We describe a method for multiplex detection of mutations in which the solid-phase minisequencing principle is applied to an oligonucleotide array format. The mutations are detected by extending immobilized primers that anneal to their template sequences immediately adjacent to the mutant nucleotide positions with single labeled dideoxynucleoside triphosphates using a DNA polymerase. The arrays were prepared by coupling one primer per mutation to be detected on a small glass area. Genomic fragments spanning nine disease mutations, which were selected as targets for the assay, were amplified in multiplex PCR reactions and used as templates for the minisequencing reactions on the primer array. The genotypes of homozygous and heterozygous genomic DNA samples were unequivocally defined at each analyzed nucleotide position by the highly specific primer extension reaction. In a comparison to hybridization with immobilized allele-specific probes in the same assay format, the power of discrimination between homozygous and heterozygous genotypes was one order of magnitude higher using the minisequencing method. Therefore, single-nucleotide primer extension is a promising principle for future high-throughput mutation detection and genotyping using high density DNA-chip technology.
我们描述了一种多重检测突变的方法，该方法将固相微测序原理应用于寡核苷酸阵列形式。通过使用DNA聚合酶，用单标记的双脱氧核苷三磷酸延伸与紧邻突变核苷酸位置的模板序列退火的固定引物来检测突变。通过将待检测的每个突变的一个引物偶联到小玻璃区域上来制备阵列。跨越九个疾病突变的基因组片段被选为检测靶点，在多重PCR反应中进行扩增，并用作引物阵列上微测序反应的模板。通过高度特异性的引物延伸反应，在每个分析的核苷酸位置明确界定了纯合和杂合基因组DNA样本的基因型。与在相同检测形式下与固定的等位基因特异性探针杂交相比，使用微测序方法区分纯合和杂合基因型的能力高一个数量级。因此，单核苷酸引物延伸是未来使用高密度DNA芯片技术进行高通量突变检测和基因分型的一种有前景的原理。
