Comparison across grinding and broaching methods of DNA extraction from dental pulp

Abstract

Introduction

Various methods are available for obtaining yield of High Molecular Weight DNA for subsequent Multiplex PCR from teeth sample. However, a proper validation and comparison of these methods for obtaining maximum output with quantifiable output not clearly established. The primary objective of this study was to measure the effectiveness across the grinding and broaching methods of DNA extraction in freshly extracted tooth pulp, its quality check using Real Time-Polymerase Chain Reaction (RT-PCR) and subsequent multiplex STR typing.

Method

A total of 40 freshly extracted normal teeth were randomly collected. Isolation and extraction of DNA was done by organic extraction method. Precipitation of samples was done using 100% chilled ethyl alcohol followed by concentration and washing of DNA via column- based technique using DNA binding buffer and DNA wash buffer. Agarose gel electrophoresis was done to roughly estimate the DNA content while exact quantity of DNA was estimated by RT-PCR technique.

Result

Independent sample t test analysis revealed that the mean quantity of DNA (in μg/l) was significantly higher in broaching method (M=29.91, SD=0.65) than grinding (M=9.71, SD=0.45), t (38) =114.19, p<0.000. Similarly, quality of DNA was analyzed using smear quality and it was found that the quality of DNA for broaching (M=4.55, SD=0.51) was significantly higher that the grinding method (M=2.55, SD =0.6), t (38) =11.3, p<0.000. As far as multiplex STR typing was concerned, a more clear, sharp and balanced genotype plot was obtained from the DNA obtained via broaching method in comparison to grinding method.

Conclusion

The significant quantitative and qualitative loss of DNA was observed in tooth samples processed via grinding method compared to broaching method which was further supported by the fact that DNA profiles generated from the DNA extracted using broaching method provided adequate resolution of the autosomal markers and sex identification marker (amelogenin marker) in the present study which is valuable for human identification and the gender identification finally leading to individualization.