Effect of degenerate oligonucleotide primed PCR primer designs

The production of genomic DNA libraries for metabolic engineering and biological discovery applications has been simplified through the use of degenerate oligonucleotide primed PCR (DOP-PCR). This procedure can perform whole genome amplification and result in gene-size DNA fragments that clone with very high efficiency into commercial linearized T-tailed vectors. This research focused on the design of primers for DOP-PCR that can produce libraries of adequate DNA fragment size while maintaining full genome coverage and minimizing bias towards amplifying only certain regions of a genome. The basic primer design consists of a static 5’ region, followed by a guanine (“G”) rich region (6-9 residues), and a degenerate (equal probability of A/T/G/C) region of 3-9 residues. Thermodynamics were employed to design primer pools so each combination of degenerate region could exist without being confined by a hairpin structure. Primers were tested by DOP-PCR amplification of 3 genomes: E. coli NEB 10-beta (50.8% GC content), Clostridium acetobutylicum ATCC 824 (30.9% GC), and Comamonas testosteroni ATCC 11996 (618% GC). Results from 15 primer candidates and all genomes were analyzed by next-generation sequencing.