5 years ago

Improved DOP-PCR (iDOP-PCR): A robust and simple WGA method for efficient amplification of low copy number genomic DNA

Tatiana V. Kramarova, Konstantin B. Ignatov, Simon C. Baker, Vera V. Ustinova, Dmitriy S. Shcherbo, Vladimir M. Kramarov, Andrew A. Shelenkov, Alexey V. Garkovenko, Maria R. Tokarenko, Konstantin A. Blagodatskikh, Ekaterina V. Barsova

by Konstantin A. Blagodatskikh, Vladimir M. Kramarov, Ekaterina V. Barsova, Alexey V. Garkovenko, Dmitriy S. Shcherbo, Andrew A. Shelenkov, Vera V. Ustinova, Maria R. Tokarenko, Simon C. Baker, Tatiana V. Kramarova, Konstantin B. Ignatov

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.

Publisher URL: http://journals.plos.org/plosone/article

DOI: 10.1371/journal.pone.0184507

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