title: 16S rDNA v12 Subject 1 Mucosa
organism: human metagenome
description: Subjects were enrolled via a protocol approved by the University of Connecticut Health Center Institutional Review Board. Criteria for inclusion of subjects included being 21 years of age or older and willing and able to provide informed consent. No subject had been diagnosed with a systemic disease or was regularly taking any medication except multivitamin supplements. All subjects had at least 25 teeth and were in good oral health, defined by the absence of mucosal disease, no visible caries lesions or periodontal disease defined by a Community Periodontal Index of Treatment Needs greater or equal to 2 in any sextant of the mouth, and with all teeth present evaluated at six sites. Additionally, no subject had taken systemic antibiotics within 2 months before sampling or used commercial probiotic supplements (greater than or equal to 108 organisms per day). Subjects were instructed not to perform any oral hygiene habits for 4 hours prior to sampling. A mucosal swab sample was collected by passing a single CatchAllTM swab through the entire area of the right and left buccal mucosa for a 10 second period. The swab was immediately swirled in a tube containing 500 uL of TE buffer (20 mM Tris pH 7.4, 2 mM EDTA) and pressed against the tube walls to transfer the material to the solution, which was stored at -80°C. DNA was isolated by mixing the TE-resuspended sample with lysozyme (final concentration of 20 mg mL-1) followed by incubation at 37°C for 30 minutes. This was followed by addition of buffer AL and Proteinase K (final concentration 1.23 mg mL-1) and incubation at 56°C overnight. Samples were then incubated at 95°C for 5 minutes and DNA isolated using a commercially available kit according to the instructions of the manufacturer (Qiagen, Valencia, CA; DNeasy Blood and Tissue kit). DNA was eluted in MD5 solution (MoBio Laboratories, Carlsbad, CA).