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

Author(s)

Thais Francini GARBIERI ; Daniel Thomas BROZOSKI ^[2] ; Thiago José DIONÍSIO ^[3] ; Carlos Ferreira SANTOS ^[4] ; Lucimara Teixeira das NEVES

• Document type: Journal article
• Source: Journal of Applied Oral Science; v. 25, n. 2, p. 147-158, 2017-04-00.

Abstract

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. (AU)

• FAPESP's process: 11/22434-7 - Different methods for DNA extraction associated with different storage periods of total saliva in order to obtain genomic DNA for genetic sequencing
• Grantee: Thais Francini Garbieri
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 09/53848-1 - Acquisition of two large-capacity devices (a Milliplex Analyzer Xponent 3 and accessories and a 7900 HT Fast Real Time PCR System) for research conductedat the University of São Paulo School of Dentistry and at other research institutes
• Grantee: Carlos Ferreira dos Santos
• Support Opportunities: Multi-user Equipment Program