Abstract
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.
MeSH terms
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DNA Copy Number Variations
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DNA Primers*
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Gene Dosage*
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Gene Library
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Genome, Human*
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Genomics* / methods
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Genotype
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High-Throughput Nucleotide Sequencing
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Humans
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Polymerase Chain Reaction / methods*
Grants and funding
The work was supported by the Ministry of Education and Science of Russian Federation, grant 14.579.21.0012 (ID # RFMEFI57914X0012) and by the grant 0112-2016-0006 from the Russian State Budget. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Genetic Expertise LLC provided support in the form of salaries for A.V.G. and provided equipment for STR-analysis, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Syntol JSC provided support in the form of salaries for V.V.U. and reagents for NGS-analysis, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Bioline Ltd provided support in the form of salaries for S.C.B., but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.Evrogen JSC provided equipment for NGS-analysis and provided support in the form of salaries for authors E.V.B., D.S.S., A.A.S. and M.R.T., but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.