Human DNA extraction from whole saliva that was fresh or stored for 3, 6 or 12 months using five different protocols

Abstract Saliva when compared to blood collection has the following advantages: it requires no specialized personnel for collection, allows for remote collection by the patient, is painless, well accepted by participants, has decreased risks of disease transmission, does not clot, can be frozen before DNA extraction and possibly has a longer storage time. Objective and Material and Methods This study aimed to compare the quantity and quality of human DNA extracted from saliva that was fresh or frozen for three, six and twelve months using five different DNA extraction protocols: protocol 1 – Oragene™ commercial kit, protocol 2 – QIAamp DNA mini kit, protocol 3 – DNA extraction using ammonium acetate, protocol 4 – Instagene™ Matrix and protocol 5 – Instagene™ Matrix diluted 1:1 using proteinase K and 1% SDS. Briefly, DNA was analyzed using spectrophotometry, electrophoresis and PCR. Results Results indicated that time spent in storage typically decreased the DNA quantity with the exception of protocol 1. The purity of DNA was generally not affected by storage times for the commercial based protocols, while the purity of the DNA samples extracted by the noncommercial protocols typically decreased when the saliva was stored longer. Only protocol 1 consistently extracted unfragmented DNA samples. In general, DNA samples extracted through protocols 1, 2, 3 and 4, regardless of storage time, were amplified by human specific primers whereas protocol 5 produced almost no samples that were able to be amplified by human specific primers. Depending on the protocol used, it was possible to extract DNA in high quantities and of good quality using whole saliva, and furthermore, for the purposes of DNA extraction, saliva can be reliably stored for relatively long time periods. Conclusions In summary, a complicated picture emerges when taking into account the extracted DNA’s quantity, purity and quality; depending on a given researchers needs, one protocol’s particular strengths and costs might be the deciding factor for its employment.


Introduction
For large scale genetic studies, the amount and quality of DNA available from a sample is an essential requirement. Usually the preferred source for the collection of genetic material for these studies is peripheral blood 1 because it yields large amounts of per mL of blood) 16  In general, molecular analysis requires several processing steps 2 with DNA extraction being one of the most important steps for the success of a molecular genetic study 13 . All the reasons stated above have led to searches for alternative methods to obtain genetic material for studies requiring DNA, with saliva being considered one of the best candidates 13 .
B r i e f l y, t h e s u b l i n g u a l , p a r o t i d a n d t h e submandibular glands secrete saliva. Furthermore, in human oral mucosa that occurs approximately every 2.7 hours 5 ultimately leads to saliva composed of ~75% epithelial cells (~430,000 cells per mL 5 ) and ~25% leukocytes (2 to 136,000 cells per mL) 7,19 depending on the oral health of the individual. Endler, et al. 7 (1999) found on average, at least 58% of the epithelial cells present in collected saliva samples to be viable with intact genomic DNA 7 . However, their extraction protocol, based on Sambrook, et al. 17 (1988), typically extracted the majority of DNA from leukocytes and they hypothesized that their protocol more easily extracted DNA from leukocytes compared to epithelial cells in their saliva samples from bone marrow transplant patients 17 . However, it remains to be studied if modern DNA extraction protocols differ in this respect from the protocol based on Sambrook, et al. 17 (1988). Dawes (2003), moreover, found that saliva from his volunteers typically contained ~430,000 epithelial cells per mL, and that, on average, each epithelial cell had approximately 80 to 100 bacteria attached 5  protocol included collecting saliva without a suspension buffer 1 . Whole saliva was centrifuged in a 1.5 mL microcentrifuge tube at 10,000 g for 5 minutes.
The supernatant was discarded and the pellet was       A study by Goode, et al. 8 (2014) details an optimized procedure for extracting DNA using reagents from the Puregene extraction kit (Qiagen) 8 . They found that using a reagent volume smaller then recommend by the manufacturer did not compromise the amount of DNA extracted and optimized costs. Notably, their protocol is similar to protocol 3 used in this study.

Results
Further examination may reveal that costs and DNA extractions can be further optimized in a similar manner.
A search of the literature revealed only one DNA extraction from microorganisms from cultured mediums using the Instagene matrix protocol 3, 10 . This study noted the successful extraction of DNA from this with the present study where the extractions were from whole saliva.

Conclusion
When viewed with the perspective gained from this study and from other independent studies, it is estimated that commercial kits, sometimes independent of the storage time, provide consistent results in terms of the concentration and purity of DNA extracted from whole saliva, especially when dealing with saliva that has been frozen and/or stored for a long time. If whole genomic DNA is needed, then only protocol 1 can be recommended. The less expensive with stored frozen saliva; however, fresh saliva or saliva stored for short durations might be adequate albeit fragmented. Although, as stressed above, a complicated picture emerges when taking into account the extracted DNA's quantity, purity, quality and the protocols ability to provide decent starting material for PCR, and, depending on a given researchers needs, one protocol's particular strengths and costs might be the deciding factor for its employment.