Methods for ensuring the highest DNA concentration and yield in future and retrospective trace DNA extracts.
Author(s): Dilley K, Pagan F, Chapman B
Publication: Sci Justice, 2021, Vol. 61, Page 193-197
PubMed ID: 33736853 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to evaluate the effects of elution volume and successive elution, and to compare three different concentration filters on DNA recovery and concentration from saliva aliquots with low DNA content.
Conclusion of Paper
Slightly less than 50% of DNA was recovered from saliva using the QIAamp DNA Investigator kit; similar DNA yields were achieved with elution volumes of 50 and 100 µL, although DNA concentration was two-fold higher when the elution volume was 50 µL compared to 100 µL. When successive elution steps were performed, mean DNA recovery increased to 63%. Spin columns increased DNA concentration of samples with an elution volume of 100 µL, although some DNA loss was observed with each of the three columns. While the highest DNA concentration was achieved using the Vivaspin column (0.376 ± 0.072 ng/µL), the recovery of input DNA was slightly higher with the MicroSep, than the Vivaspin or Amicon filters (77% versus 68% and 56%, respectively).
Studies
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Study Purpose
The purpose of this study was to evaluate the effects of elution volume and successive elutions, and to compare three different concentration filters on DNA recovery and concentration from saliva aliquots with low DNA content collected from a single donor. Saliva was obtained from one male volunteer by vigorously rinsing his mouth with saline. Cells were enumerated using a hemocytometer. Saliva was aliquoted based on an estimated fixed amount of DNA, i.e. if each cell contained 6 pg DNA, each 15 µL aliquot of saliva would contain ~6.3 ng DNA. DNA was extracted from each aliquot using the QIAamp DNA Investigator kit and eluted with 50 or 100 µL ATE buffer or aliquoted twice with 50 µL ATE with carrier RNA added. For aliquots eluted with 100 µL buffer, potential effects of DNA concentration with the Amicon Ultra-0.5 30 K, Microsep Advance 30 K or Vivaspin 500 30 K filter and their respective centrifugation conditions were investigated filters. DNA was quantified using the real-time PCR-based ThermoFisher Quantifiler Trio DNA Quantification Kit.
Summary of Findings:
On average, 48% and 49% of DNA was recovered from saliva aliquots using the QIAamp DNA Investigator kit when eluted with 50 and 100 µL, respectively. Importantly, since the same amount of DNA was recovered, elution with 50 µL resulted in a two-fold higher DNA concentration than elution with 100 µL. When successive elution steps (50 µL twce) were performed and carrier RNA was added, mean DNA recovery increased to 63% and was significantly higher than when a single 100 µL elution was performed (P=0.03). Spin columns increased DNA concentration of samples with a 100 µL elution volume, with the highest concentration achieved using the Vivaspin column (0.376 ± 0.072 ng/µL) and the lowest using the Amicon filter (0.079 ± 0.012 ng/µL), but the final sample volume was also lowest using the Vivaspin column. Each column resulted in some DNA loss, but the highest recovery of the DNA relative to the DNA input amount was achieved with the MicroSep Filter (77%, 2.305±0.615 ng) followed by Vivaspin (68%, 2.097±0.386 ng) and the Amicon filter (56%, 1.717±0.254 ng).
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte purification Amicon Ultra-0.5 30 K
Vivaspin 500 30 K
Microsep Advance 30 K
Analyte Extraction and Purification Rehydration of dried sample/specimen Twice with 50µL ATE buffer with carrier RNA
50µL ATE buffer
100µL ATE buffer