Template length, concentration and guanidine and cytosine content influence on multiple displacement amplification...

Detection of low abundance human health pathogens in environmental samples is a challenge for water monitoring. This limitation can be overcome by the introduction of multiple displacement amplification (MDA) where a minute amount of genetic material can be amplified using a phi-29 DNA polymerase. However, the genetic makeup and the concentration of the polynucleotides might influence the amplification process due to inherent assay bias. Herein, a series of experiments were designed to demonstrate the effect of genome length, guanidine and cytosine content, and template concentration on the efficiency of MDA. Quantitative polymerase chain reaction (qPCR) was performed to quantify pre- and post-MDA concentrations of selected genes. Linear regression between pre- and post-MDA log gene copies L^-1 of both environmental and lab-grown samples showed a positive correlation (F = 77.59, P < 0.001, R² = 0.7, slope = 1.01). Correlation between relative polynucleotide increase after MDA and target organism length and gene target guanidine and cytosine (G + C) content (F = 4.3, P = 0.02) shows that lower G + C and higher genome length is favored in the MDA process. The MDA process was shown to favor a longer genome over a shorter genome (1.19 and 1.04 change in log gene copy L^-1, respectively) and a lower G + C content over a higher G + C content (1.11 and 0.61 change in log gene copy L^-1, respectively). There was no MDA bias observed when polynucleotides had the same G + C and genome length but different initial concentrations. This study highlights the need for increased caution when interpreting relative abundance of organisms amplified by MDA such as in next generation sequencing.