Improved multiple displacement amplification with φ29 DNA polymerase for genotyping of single human cells
Improved multiple displacement amplification with φ29 DNA polymerase for genotyping of single human cells
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DOI：
10.2144/000112755
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发表时间：
2008-06-01
期刊：
影响因子：
2.7
通讯作者：
Vidali, Andrea
中科院分区：
文献类型：
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作者：
Kumar, Gyanendra;Garnova, Elena;Vidali, Andrea
对单细胞多个基因座进行基因分型的能力对于肿瘤发生、减数分裂、受精和胚胎发生等细胞过程的研究具有显着的益处。我们报告了一种简单的两步单管方案，使用 GenomiPhi V2 DNA 扩增试剂盒的组件对单个人类细胞进行全基因组扩增 (WGA)。我们首次证明，在 4 小时内，以最少量的人工 DNA 合成，从单个人类细胞中可靠地生成 4-7 μg 扩增 DNA。对从单细胞扩增的 DNA 进行 13 个杂合短串联重复序列 (STR) 和 7 个杂合单核苷酸多态性 (SNP) 的基因分型，并将基因分型结果与纯化的基因组 DNA 进行比较。 STR 的基因分型准确度（对任何特定 STR 或 SNP 进行准确基因分型的单细胞扩增的百分比）从 37% 到 100%（平均为 80%）不等，对于 SNP 则从 89% 到 100%（平均为 94%）不等。我们认为本报告中描述的方法适用于单细胞 WGA，其产品随后可用于许多应用，例如植入前遗传分析 (PGD)。
The ability to genotype multiple loci of single cells would be of significant benefit to investigations of cellular processes such as oncogenesis, meiosis, fertilization, and embryogenesis. We report a simple two-step, single-tube protocol for whole-genome amplification (WGA) from single human cells using components of the GenomiPhi V2 DNA Amplification kit. For the first time, we demonstrate reliable generation of 4-7 mu g amplified DNA from a single human cell within 4 h with a minimum amount of artifactual DNA synthesis. DNA amplified from single cells was genotyped for 13 heterozygous short tandem repeats (STRs) and 7 heterozygous single nucleotide polymorphoisms (SNPs), and the genotyping results were compared with purified genomic DNA. Accuracy of genotyping (percent of single-cell amplifications genotyped accurately for any particular STR or SNP) varied from 37% to 100% (with an average of 80%) for STRs and from 89% to 100% (averaging 94%) for SNPs. We suggest that the method described in this report is suitable for WGA from single cells, the product of which can be subsequently used for many applications, such as preimplantation genetic analysis (PGD).