The impact of different DNA extraction methods on the analysis of microbial diversity of oral saliva from healthy youths by polymerase chain reaction-denaturing gradient gel electrophoresis.

BACKGROUND/PURPOSE: Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), as a conventional molecular technique, was utilized to analyze the diversity of oral microbiota. However, studies found that the results of PCR-DGGE were affected by the DNA isolation method. This study compared QIAamp DNA Micro Kit extraction method with the phenol and chloroform extraction method for DNA isolation of saliva of healthy youths and analyzed PCR-DGGE fingerprints.

MATERIALS AND

METHODS

In the first stage, samples were divided into two after collection from eight health youths. Two methods were used to isolate the DNA for PCR-DGGE analysis. In the second stage, another 16 samples were collected from 14 youths. The better method, QIAamp DNA Micro Kit, was used to isolate the DNA for PCR-DGGE analysis. The cluster analysis was performed with unweighted pair-group method with arithmetic means.

RESULTS

The results in the first stage showed that the QIAamp DNA Micro Kit extraction method was more suitable for DNA extraction of saliva than the phenol-chloroform extraction method. In the second stage, the bands were changed into numbers "0", "1", and "2" to analyze the similarity of samples according to the bands' lightness. The similarity indices of different periods from the same individual showed that the microbiological composition was very similar (>0.95), while those from different individuals varied greatly (<0.90).

CONCLUSION

PCR-DGGE was more accurate in assessing oral microbial diversity by QIAamp DNA Micro Kit. Different individuals had large differences in oral microbial diversity but also had some common microbial dominant communities.