The Effects of Storage of Human Saliva on DNA Isolation and Stability

The most important progress in diagnostic sciences is the increased sensitivity and specificity in diagnostic procedures due to the development of micromethodologies and increasing availability of immunological and molecular biological reagents. The technological advances led to consider the diagnostic use of saliva for an array of analytes and DNA source. The purpose of the present study was to compare DNA from saliva with those from blood and buccal swab, to evaluate diagnostic and forensic application of saliva, to investigate the changes of genomic DNA in saliva according to the storage temperature and period of saliva samples, and to evaluate the integrity of the DNA from saliva stored under various storage conditions by PCR analysis. Peripheral venous blood, unstimulated whole saliva, stimulated whole saliva, and buccal swab were obtained from healthy 10 subjects (mean age: 29.9 ± 9.8 years) and genomic DNA was extracted using commercial kit. For the study of effects of various storage conditions on genomic DNA from saliva, stimulated whole saliva were obtained from healthy 20 subjects (mean age: 32.3 ± 6.6 years). After making aliquots from fresh saliva, they were stored at room temperature, 4℃, -20℃, and -70℃. Saliva samples after lyophilization and dry-out procedure were stored at room temperature. After 1, 3, and 5 months, the same experiment was performed to investigate the changes in genomic DNA in saliva samples. In case of saliva aliquots stored at room temperature and dry-out samples, the results in 2 weeks were also included. Integrity of DNA from saliva stored under various storage conditions was also evaluated by PCR amplification analysis of s-globin gene fragments (989-bp). The results were as follows: 1. Concentration of genomic DNA extracted from saliva was lower than that from blood (p＜0.05), but there were no significant differences among various types of saliva samples. Purities of genomic DNA extracted from stimulated whole saliva and lyophilized one were significantly higher than that from blood (p＜0.05). Purity of genomic DNA extracted from buccal swab was lower than those from various types of saliva samples (p＜0.05). 2. Concentration of genomic DNA from saliva stored at room temperature showed gradual reduction after 1 month, and decreased significantly in 3 and 5 months (p＜0.05, p＜0.01, respectively). Purities of DNA from saliva stored for 3 and 5 months showed significant differences with those of fresh saliva and stored saliva for 1 month (p＜0.05). 3. In the case of saliva stored at 4℃ and -20℃, there were no significant changes of concentration of genomic DNA in 3 months. Concentration of DNA decreased significantly in 5 months (p＜0.05). 4. There were no significant differences of concentration of genomic DNA from saliva stored at -70℃ and from lyophilized one according to storage period. Concentration of DNA showed decreasing tendency in 5 months. 5. Concentration of genomic DNA immediately extracted from saliva dried on Petri dish were 60% compared with that of fresh saliva. Concentration of DNA from saliva stored at room temperature after dry-out showed rapid reduction within 2 weeks (p＜0.05). 6. Amplification of s-globin gene using PCR was successful in all lyophilized saliva stored for 5 months. At the time of 1 month, s-globin gene was successfully amplified in all saliva samples stored at -20℃ and -70℃, and in some saliva samples stored at 4℃. s-globin gene was failed to amplify in saliva stored at room temperature and dry-out saliva.