148 - Novel Methods for DNA Extraction from Carious Primary Teeth

Abstract:


Purpose: To develop a method for isolation of DNA from Formalin-Fixed Paraffin-Embedded (FFPE) and Formalin-Fixed, Macro-dissected, and Pulverized (FFMP) carious primary teeth for the ultimate purpose of microbiome assessment.


Methods: Twenty carious primary teeth were collected from 20 children ages 3 to 13 years old and fixed in 10% formalin. Teeth processed by FFMP were macro-dissected at 200-500µm thickness using an IsoMet precision saw. Sections were cryogenically cooled with liquid nitrogen, pulverized into powder in a stainless-steel tube, and bead-beated using glass beads. Teeth processed by FFPE were decalcified in formic acid for 14 days, paraffin embedded and sectioned across a 100µm thickness. DNA was extracted using QIAGEN DNA isolation kits. Overall DNA yield was analyzed spectrophotometrically. Microbial DNA was quantified using PCR with fungal and bacterial primers.


Results: Thin sections of carious primary teeth were reliably created by FFMP and FFPE processed teeth. DNA was consistently extracted from both. There was no significant difference in the average DNA quantity isolated from FFMP and FFPE samples (P>.05, t-test). The average bacterial and fungal DNA isolated from FFMP and FFPE samples was comparable (P>.05, t-test).


Conclusions: This study establishes two novel methods that demonstrate feasible and consistent microbial DNA isolation from FFMP and FFPE carious primary teeth. Formalin fixation, decalcification, and paraffin embedding do not appear to decrease the DNA yield. No statistically significant difference in DNA yield was observed between FFMP and FFPE methods.

Identify Supporting Agency and Grant Number: Research supported by Paul K. Losch Fund and N. Sue Seale Pediatric Dentistry Awards.