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

Autor(es)

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

• Tipo de documento: Artigo Científico
• Fonte: Journal of Applied Oral Science; v. 25, n. 2, p. 147-158, 2017-04-00.

Resumo

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)

• Processo FAPESP: 11/22434-7 - Diferentes métodos de extração de DNA associados a diferentes períodos de armazenamento de saliva total para obtenção de DNA genômico para sequenciamento direto
• Beneficiário: Thais Francini Garbieri
• Modalidade de apoio: Bolsas no Brasil - Iniciação Científica
• Processo FAPESP: 09/53848-1 - EMU: Aquisição de dois equipamentos de grande porte (Milliplex Analyzer Xponent 3 e acessórios e 7900 HT Fast Real Time PCR System e acessórios) para a realização de pesquisas por pesquisadores de diversos departamentos da FOB-USP e de outras instituições de pesquisa
• Beneficiário: Carlos Ferreira dos Santos
• Modalidade de apoio: Auxílio à Pesquisa - Programa Equipamentos Multiusuários