AUTHOR=Martino Amanda , Rhodes Matthew E., Biddle Jennifer F., Brandt Leah D., Tomsho Lynn P., House Christopher H. TITLE=Novel Degenerate PCR Method for Whole-Genome Amplification Applied to Peru Margin (ODP Leg 201) Subsurface Samples JOURNAL=Frontiers in Microbiology VOLUME=Volume 3 - 2012 YEAR=2012 URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2012.00017 DOI=10.3389/fmicb.2012.00017 ISSN=1664-302X ABSTRACT=
A degenerate polymerase chain reaction (PCR)-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. While optimized here for use with Roche 454 technology, the general framework presented may be applicable to other next generation sequencing systems as well (e.g., Illumina, Ion Torrent). The method, which we have called random amplification metagenomic PCR (RAMP), involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3′ end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured