SNP genotyping on a genome-wide amplified DOP-PCR template
SNP genotyping on a genome-wide amplified DOP-PCR template
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DOI：
10.1093/nar/gnf125
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发表时间：
2002-11-15
影响因子：
14.9
通讯作者：
Tolle, R
中科院分区：
文献类型：
--
作者：
Grant, SFA;Steinlicht, S;Tolle, R
随着对高通量单核苷酸多态性（SNP）基因分型的需求不断增加，基因组DNA的数量往往低于所需的检测数量。我们研究了使用退化寡核苷酸引物聚合酶链反应（dopp - pcr）为我们的荧光极化模板定向染料终止物掺入检测的SNP基因分型方法生成模板。dopp - pcr采用简并引物（5'-CCGACTCGAGNNNNNNATGTGG-3'）对DNA进行非特异性均匀扩增。该方法已成功应用于微卫星基因分型。我们比较了dopp - pcr扩增的基因组DNA与作为模板的基因组DNA的基因分型。结果从可行性、等位基因丢失、基因分型准确性和高通量基因分型环境下的储存条件等方面进行了分析。dopp - pcr获得了总体满意的结果，但在基因型分配的准确性和质量上有一定的损失。从dopp - pcr模板生成的基因型的准确性和质量也取决于储存条件。将载体DNA加入到最终浓度为10 ng/ ml的浓度中，改善了结果。总之，我们已经成功地使用dopp - pcr扩增我们的基因组DNA收集，用于随后的SNP基因分型作为标准过程。
With the increasing demand for higher throughput single nucleotide polymorphism (SNP) genotyping, the quantity of genomic DNA often falls short of the number of assays required. We investigated the use of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) to generate a template for our SNP genotyping methodology of fluorescence polarization template-directed dye-terminator incorporation detection. DOP-PCR employs a degenerate primer (5'-CCGACTCGAGNNNNNNATGTGG-3') to produce non-specific uniform amplification of DNA. This approach has been successfully applied to microsatellite genotyping. We compared genotyping of DOP-PCR-amplified genomic DNA to genomic DNA as a template. Results were analyzed with respect to feasibility, allele loss of alleles, genotyping accuracy and storage conditions in a high-throughput genotyping environment. DOP-PCR yielded overall satisfactory results, with a certain loss in accuracy and quality of the genotype assignments. Accuracy and quality of genotypes generated from the DOP-PCR template also depended on storage conditions. Adding carrier DNA to a final concentration of 10 ng/mul improved results. In conclusion, we have successfully used DOP-PCR to amplify our genomic DNA collection for subsequent SNP genotyping as a standard process.