Blagodatskikh Konstantin A, Kramarov Vladimir M, Barsova Ekaterina V, Garkovenko Alexey V, Shcherbo Dmitriy S, Shelenkov Andrew A, Ustinova Vera V, Tokarenko Maria R, Baker Simon C, Kramarova Tatiana V, Ignatov Konstantin B
All-Russia Institute of Agricultural Biotechnology, Russian Academy of Sciences, Moscow, Russia.
Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia.
PLoS One. 2017 Sep 11;12(9):e0184507. doi: 10.1371/journal.pone.0184507. eCollection 2017.
Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.
全基因组扩增（WGA）技术用于低拷贝数DNA的非特异性扩增，特别是用于单细胞基因组和转录组扩增。多年来已经开发了许多WGA方法。一个例子是简并寡核苷酸引物PCR（DOP-PCR），它是一种非常简单、快速且廉价的WGA技术。尽管DOP-PCR被认为是WGA的开创性方法之一，但与更现代的技术相比，它只能提供低基因组覆盖率和高等位基因缺失率。在这里，我们描述了一种改进的DOP-PCR（iDOP-PCR）。我们通过使用具有强链置换活性的新型耐热DNA聚合酶（SD聚合酶）并调整引物设计，对经典的DOP-PCR进行了改进。我们比较了iDOP-PCR、经典DOP-PCR和成熟的PicoPlex技术对高拷贝数和低拷贝数人类基因组DNA进行全基因组扩增的效果。通过分析短串联重复基因型和NGS数据对扩增的DNA文库进行评估。总之，与其他测试的WGA方法相比，iDOP-PCR提供了质量更好的扩增DNA文库，尤其是当使用少量基因组DNA作为输入材料时。
