Extraction of mitochondrial DNA from tooth dentin: application of two techniques

Mitochondrial DNA (mtDNA) is a hereditary material located in mitochondria and is normally maternally inherited. Mutational analysis performed on mtDNA proved that the mutations are closely related with a number of genetic illnesses, besides being exploitable for forensic identification. Those findings imply the importance of mtDNA in the scientific field. MtDNA can be found in abundance in tooth dentin where it is kept protected by the enamel, the hardest outer part of the tooth. In this study, two techniques of mtDNA extraction were compared to determine the efficacy between the two techniques. Teeth used for the study was collected from Dental Clinic, Hospital Universiti Sains Malaysia. After the removal of tooth from the tooth socket of the patient, the tooth was kept at -20C until use. Later, pulp tissue and enamel was excised using dental bur and only the root dentin was utilized for the isolation of mtDNA by crushing it mechanically into powdered form. MtDNA was extracted using the two published methods, Pfeifer and Budowle and then subjected to spectrophotometry DNA quantification and purity, Polymerase chain reaction (PCR) amplification of hypervariable-two region of mtDNA, followed by DNA sequencing to analyze the reliability of the extraction techniques. In conclusion, both techniques proved to be efficient and capable for the extraction of mtDNA from tooth dentin.

Telomerase activity in Malaysian patients with central nervous system tumors.

Telomerase, the enzyme that stabilizes telomere length is reactivated with almost all cancer types, and may be a useful diagnostic marker for malignancy. Telomerase activity has been detected in germ line cells and most cancer cells, whereas most normal somatic cells have no clearly detectable telomerase activity. In our study, we aim to detect telomerase activity in 20 human central nervous system tumors from Malaysian patients. Telomerase activity was detected based on a highly sensitive procedure consisting of a CHAPS detergent-based extraction from frozen tissues and a PCR-based telomeric repeat amplification protocol (TRAP) using a TRAPEZE Telomerase Detection Kit (Intergen, Co). Telomerase activity was considered positive when a ladder of products was observed starting at 50bp, with 6bp increments. The activity was detected in 30% of the samples analysed, included glioblastoma multiforme, meduloblastoma, paraganglioma and oligodendroglioma. The result of Fisher's exact test indicated that there was a significant association between telomerase activity status with tumor grade (p=0.003). These results suggest that telomerase activity may be an important marker for tumor malignancy.