INSIGHTS INTO THE RARE BIOSPHERE: EXHAUSTIVE ELIMINATION OF TOTAL DNA FROM COMPLEX MICROBIAL COMMUNITIES ENHANCES PCR RESOLUTION OF ALPHA-DIVERSITY.
Open Access
- Author:
- Chopra, Arsh
- Area of Honors:
- Science
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Charles Raymond Fisher Jr., Thesis Supervisor
Dr. Ronald Albert Markle, Thesis Honors Advisor
Jennifer Macalady, Faculty Reader - Keywords:
- PCR
PMA
Bias - Abstract:
- Use of the Polymerase Chain (PCR) reaction for the amplification of the 16S rRNA gene (rDNA) has allowed the cultivation-independent interrogation of complex microbial communities for taxon richness. There are many well-documented caveats with this technique that suggest universal underrepresentation of total prokaryotic diversity. Here we propose a novel approach aimed at increasing the taxon resolution of PCR by exhaustive amplification inhibition of template DNA from complex communities via stochastic binding of Propidium Monoazide (PMA). PMA is a DNA intercalating molecule capable of forming covalent cross-linkages to organic moieties upon light exposure used to debar template PCR amplification by prohibiting denaturation during PCR cycling. Stochastic intercalating of PMA, as opposed to exhaustive dilution, is shown to disproportionally inactivate DNA from different community members. Using species-specific real-time quantitative PCR on a defined DNA community control we show that high abundance phylotypes are inactivated to a greater extent than low abundance phylotypes by PMA-DNA covalent bond formation. To our knowledge, this is the first study to quantify the effects of template removal, via random chemical binding, on a mock DNA community using species-specific quantitative PCR. We hypothesize that the resulting higher relative proportion of low abundance taxa in the total DNA template mixture enhances amplification of rare biosphere phylotypes. Additionally, this method was applied to a complex environmental DNA sample and, following 16S rDNA PCR amplification and analysis via phylogenetic DNA microarray (PhyloChip), results indicated a 19.5% increase in taxon resolution when compared to a control. Increased taxon richness observed in the soil sample suggests the amplification of low abundance phylotypes, usually shadowed by the overwhelming presence of genetically homogenous high abundance templates, known as “rare biosphere” members. Exhaustive elimination of predominant phylotypes with PMA is a relatively simple and inexpensive method capable of increasing the resolution of existing techniques interrogating environmental taxon richness and can be readily employed to enhance the phylogenetic breadth of “at-hand” previously surveyed samples.