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 andMaterial 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.

Selection of a Noninvasive Source of Human DNA Envisaging Genotyping Assays in Epidemiological Studies: Urine or Saliva?

“…Furthermore, it is known that urinary and salivary DNA contain coextracted microbial nucleotides present in these biofluids, which could lead to an overestimation of the actual human DNA concentration measured by both of these nonspecific spectrophotometric or fluorometric methods. 23,26,40,49 The use of a real-time qPCR assay using the human-specific b-globin primers, overcomes these overestimations by determining more specifically the amount of human-intact amplifiable DNA in the total DNA extracted from urine or saliva. Our results showed that the DNA extracted from saliva contains a significant proportion of human intact DNA fragments of at least 164 bp long in contrast with the urinary DNA for which this human proportion could not be determined because it was below the detection limit for most of the samples.…”

Saliva—Friend and Foe in the COVID-19 Outbreak

“…Saliva also represents an attractive biofluid source option for the detection of SARS-CoV-2, due to being non-invasive, easy-to-access, and low-cost, as well as having the ability to "mirror" systemic and local disease status [50]. It is well-known that saliva harbors a wide range of circulatory components (Figure 2), such as pro-inflammatory cytokines [51,52], chemokines [53], matrix metalloproteinases [54,55], mitochondrial DNA [56], genomic DNA [57], bacteria [58], SARS-CoV and SARS-CoV-2 virus [30,31,59], SARS-CoV antibodies [59], miRNAs [60], and extracellular vesicles (EVs) [61]. Furthermore, saliva samples can be stored at -80 • C for several years with little degradation [62].…”

Genetic conditionality of strength by the ACE gene in women horse riders

“…The kit’s procedure was modified by the collection and isolation of saliva DNA from 1 ml of preserved saliva samples, and overnight sample incubation with proteinase K. Isolated DNA was suspended in ddH20. NorgenBiotek commercial kits, the manufacturer’s instructions were followed [17].…”

“…The 260/280 nm absorbance ratio between 1.6 and 2.0 for samples were considered pure. Ratio lower than 1.6 testifies to higher protein contaminates [17,18].…”