DGGE Profiles From Four Extraction Methods

METHODS: Pooled supragingiva plaques from both severe early childhood caries and caries free groups were collected and divided to provide replicates for each experimental protocols. Aliquots were processed using different bacterial DNA isolation which are phenol-chloroform isoamyl alcohol, CTAB and two commercially available bacterial DNA isolation kits. (Qiagen Genomic Extraction kit, USA, Nucleospin Genomic Extraction kit, Macherey-Nagel, Germany)

RESULTS: Four DNA extraction methods were compared by spectrophotometer, agarose gel electrophoresis, and denaturing gredient gel electrophoresis. All samples were standardized. Analysis of DGGE profiles, generated by polymerase chain reaction of purified DNA extracts, demonstrated that the choice of DNA extraction method significantly influenced the bacterial community profiles. There was a different in the total amount of extracted DNA (from 0.9 to 4.2 mg yielded). Moreover, each method provided different band patterns under the same dgge condition (range of denaturing gradient= 30:70, V=60, tem=50C, running time = 16 h). DNA extraction efficiency and number of bands were significantly higher (p<0.0001) using the two commercial kits compared to phenol-chloroform isoamyl alcohol and CTAB respectively (max:min=5:2). The effect of sample freezing on DNA recovery efficiency prior to DNA extraction was also examined. DNA concentrations from samples extracted within 24 hr were measured and compared to extracting after an overnight freeze.

CONCLUSION: Results indicated that the DNA efficiency and number of bands were significantly higher (p<0.0001) using two commercial kits compared to phenol-chloroform isoamyl alcohol and CTAB respectively. Moreover, freeze-thaw samples during a short storage time (no longer than 3 months) had no effect on DGGE fingerprint in this study.