Doryoku SEKIYA

A Novel DNA-Capturing method for Correlative Explanation of Function and Phylogeny of Environmental Microorganisms.

Hiroyuki IMACHI, Akiyoshi OHASHI, Hideki HARADA

The number of microorganisms that have been successfully identified and isolated represents only a small portion of the total that is believed to exist. To analyze uncultivated microbial consortia, analyses based on 16S rRNA or other genes were widely employed, which can describe the diversity of phylogenies genes or functional of microorganisms. However, it is difficult to link functional genes and phylogenies, which could be helpful for comprehensive explanation of the microorganism. To solve the problem we proposed a novel methodology for analyzing microorganisms in this study. First, capturing a genomic DNA with mega base pairs (Mb) length specifically from microbial consortia. Secondly the captured genomic DNA was analyzed based on both functional genes and the 16S rRNA gene. The keys of the DNA-capturing method as followes; 1) specificity, 2) length (e.g. Mega base pairs) 3) high recovery from low abundance of targeted DNA. However there is no report about DNA capturing methods satisfying the keys. Therefore we developed a novel DNA capturing method for linking functions and phylogenies of microorganisms.
For this purpose we applied Peptide nucleic acid (PNA) probes, which hybridize with their complementary nucleic acid sequence with greater affinity and specificity than the conventional DNA probe. At firstly, a protocol for DNA capturing was roughly established using PNA probes combined with streptavidine-coated magnetic beads. However, by this protocol targeted DNA as well as few non-targeted DNA unspecifically also captured. The possible cause of the problem could be the entangling of the unspecific DNA in the aggregate of magnetic beads. As a solution, streptavidine-coated tube (S.A.-coated tube) was employed instead of magnetic beads. With the implication of this method, genomic DNA with over 1.5 Mb lengths specifically was captured from DNA complex containing 10% of targeted DNA. Finally, it was concluded that the novel methodology for analyzing microorganisms could be applied to a sample, which contains over the 10% of targeted microorganisms.