Filter, heat, spin: A simple and inexpensive method for DNA preparation from freshwater for use in high-throughput molecular source tracking

Molecular source tracking (MST) can improve community health by enabling the identification of the source species of fecal bacteria contamination in waterways. However, widespread adoption of this method at a large scale is hindered by the cost of commercial extraction kits and the technical expertise required to use them. We developed a simpler, efficient, scalable, accessible, and semi-quantitative method to extract DNA from environmental water samples via heat lysis. After filtering water samples onto a polycarbonate membrane, the membrane is suspended in AE buffer, heated, and then centrifuged. The liquid supernatant is then used directly in quantitative PCR (qPCR) analysis. Our filter-heat-spin (FHS) extraction method was compared to a commercial DNA extraction kit (Qiagen DNeasy). In qPCR the traditional kit extraction yielded 2.4 times more copies/μL (on average) of the target sequence, but the FHS extraction yielded 5.1 times more DNA (on average) than the traditional extraction kit due to the higher volume of buffer used in the FHS extraction. Additionally, FHS maintained identical long-term stability as compared to the kit extraction over a five-week period. Further, the cost per FHS extraction is roughly $0.05 per sample and requires 15 minutes to complete, while the typical kit extraction is roughly $4.48 per sample and requires 50 minutes to complete. The proposed FHS extraction method allows for efficient and inexpensive water sample processing, which in turn, reduces barriers for the implementation of MST.Author summary: The study developed a simple DNA extraction method for use on environmental freshwater samples. Water samples collected from sample sites are filtered through a 0.22 micron pore size polycarbonate membrane trapping bacteria and other cells on the membrane. Next the membrane is suspended in AE (aqueous elution) buffer. The sample is then heated to 95oC for 10 minutes to lyse cells and release the DNA into the solution. To obtain the supernatant with the DNA, it is centrifuged. The samples can then be used in Molecular Source Tracking via qPCR to quantify bacteria levels or bacteria species sources in waterways. When compared to commercially available kits, this filter-heat-spin (FHS) method costs significantly less money, has fewer steps that are less prone to pipetting errors, and is quicker. The DNA has a similar yield and stability. The FHS DNA extraction method can help make Molecular Source Tracking of bacteria more viable for municipalities and researchers, especially in cases with a large number of sample sites.