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Publisher Summary This chapter demonstrates the use of whole genome amplification of DNA polymerase based on multiple displacement amplification for microbial forensic PCR/ESI-MS analysis of environmental air. It also demonstrates the utility of WGA of trace DNA specimens coupled with either Roche 454 sequencing technology or Pacific Biosciences single molecule real-time sequencing technology. Challenges remain, however. Whole genome amplification by multiple displacement amplification will amplify all DNA in a specimen, which can create challenges for detection and characterization of an agent of interest in the presence of a large amount of background. Soil samples and clinical specimens present such challenges. For example, 1 μl of human blood contains 1000 copies of the human genome so identifying a pathogen in this background would necessitate >1000 human genome projects of sequencing to find the pathogen DNA. Fortunately, there are many methods for the separation of organisms by size that can be used to separate viruses from bacteria from eukaryotic cells prior to DNA extraction and WGA. Next Gen sequencing technologies, combined with whole genome amplification, offer the forensic investigator new capabilities for the rapid microbial forensic characterization of trace and noncultivable specimens that can identify key signatures that can lead to attribution, as well as identifying bioengineered and emerging threats. |
