Rapid (30-second), equipment-free purification of nucleic acids using easy-to-make dipsticks

Abstract

The complexity of current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method to purify nucleic acids from animal, plant, viral and microbial samples using a cellulose-based dipstick. Nucleic acids can be purified by dipping in-house-made dipsticks into just three solutions: the extract (to bind the nucleic acids), a wash buffer (to remove impurities) and the amplification reaction (to elute the nucleic acids). The speed and simplicity of this method make it ideally suited for molecular applications, both within and outside the laboratory, including limited-resource settings such as remote field sites and teaching institutions. Detailed instructions for how to easily manufacture large numbers of dipsticks in house are provided. Using the instructions, readers can create more than 200 dipsticks in <30 min and perform dipstick-based nucleic acid purifications in 30 s.

Fig. 1. Nucleic acid–purifying dipstick.

The dipstick is composed of two parts: (1) a 40- to 50-mm-long wax-impregnated handle, and (2) a 2 × 6-mm nucleic acid binding zone.

Fig. 2. Rapid nucleic acid purification using dipsticks.

a, Nucleic acids are captured by dipping the dipstick into the sample until the nucleic acid binding zone of the dipstick is saturated with solution. b, Contaminating compounds are removed by dipping 5 times into the wash buffer. c, Nucleic acids are eluted by dipping 15 times directly into the amplification reaction.

Fig. 3. Preparation of the dipstick blanks.

a, The wax is melted with a colored dye to make identification of the nucleic acid binding zone easier. b, The molten wax is infused into the cellulose filter paper up to ~20 mm from the end to create the dipstick blank. c, Excess wax is removed from the dipstick blank by wiping on the edge of the Petri dish. d, A pencil line is drawn on the dipstick blank to the desired length of the nucleic acid binding zone and the excess filter paper is cut away.

Fig. 4. Rapid manufacture of dipsticks using a pasta maker.

a, The dipstick blank is placed between the edges of a folded sheet of colored paper that has been inserted into the pasta maker. b, The dipstick blank is guided through the pasta maker. c, The dipsticks are gently pulled out from the pasta maker as they are being cut. d, The dipsticks are bent to release the colored paper, which can then be pulled away from the dipsticks.

Fig. 5. Purification and detection of P. syringae.

Different amounts of P. syringae culture (1–50 µl) were added to 500 µl of extraction buffer. a, The concentration of DNA in the initial samples was determined by fluorometric assay in triplicate (n = 3). b, Dipsticks were used to purify DNA from each of the samples in five replicate purifications (n = 5), which were eluted in 1× PCR buffer. The total amount of DNA in each eluate was calculated from fluorometric DNA assay data. c, Dipsticks were used to purify the DNA from the same samples and elute it directly into a qPCR reaction with primers designed against the P. syringae genome. Purifications were performed in quadruplicate (n = 4) and qPCR quantification values (2^−Cq) were calculated. All bar graphs represent mean ± SE; individual points represent raw data values. Source data