Nucleic acid purification from plants, animals and microbes in under 30 seconds

Abstract

Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries.

Fig 1. Cellulose-based paper can be used to capture and purify nucleic acids.

(A) 1 μl of purified DNA at different concentrations (0, 0.01, 0.1, 1, or 10 ng/μl) was pipetted directly onto a Whatman No.1 disc (3 mm diameter) and then washed in 200 μl 10 mM Tris (pH 8) before adding the disc to a PCR reaction. As a control 1μl of the same DNA solutions were directly added to the PCR reactions. The band intensities achieved with cellulose discs relative to direct DNA addition appear below each band. (B) An outline of the nucleic acid purification method using Whatman No.1 discs. Tissue is ground in a 1.5 ml eppendorf tube with a plastic pestle in the presence of extraction buffer. Nucleic acids are captured by a 3-mm diameter Whatman No.1 disc. The disc is then transferred to a tube containing wash buffer for one minute to remove contaminants present in the crude extract before transferring it to the tube containing the amplification reaction. The PCR reaction is performed without removing the disc from the tube. (C) Whatman No.1 discs were immersed in an Arabidopsis thaliana leaf extract before being washed once, twice, or not at all prior to amplification. As controls, 1 μl of crude extract or water (NTC) was added directly to the PCR reaction. n.a., no amplification; NTC, no template control.

Fig 2. Cellulose-based paper is capable of purifying nucleic acids from a wide range of plant and animal tissues.

(A) Genomic DNA from leaf tissues was extracted using the cellulose disc nucleic acid purification method. Universal primers designed against the 5.8S ribosomal RNA gene were used to amplify a product by PCR from each plant species with the exception of rice in which the betaine aldehyde dehydrogenase 2 (GenBank: KU308249.1) was amplified. (B) Human whole blood was diluted 1 in 5 in an extraction buffer containing proteinase K before using the cellulose disc method to purify genomic DNA in order to amplify a fragment of the BRAF gene (UniGene Hs.550061). 1 μl of each of the raw lysates was also added directly into separate PCR reactions. Purified Hela cells genomic DNA was used as a positive control. (C) Genomic DNA purified from a human melanoma cell line (LM-MEL-70) using the cellulose disc method was used to amplify a fragment of the 28S ribosomal gene. As a control, 1 μl of the raw lysate was added directly into a separate PCR reaction. NTCs involved adding 1 μl of water instead of DNA template. BRAF, human V-raf murine sarcoma viral oncogene homolog B1; NTC, no template control.

Fig 3. DNA and RNA extraction from plant and animal pathogens using cellulose-based paper.

(A) DNA was purified from P. syringae-infected Arabidopsis leaves at different stages of infection using cellulose discs and a fragment of the P. syringae genome amplified by PCR. The band intensities achieved with cellulose discs relative to the positive control appear below each band. (B) A Whatman No.1 disc was used to purify DNA from a lung swab of a pig infected with A. pleuropneumoniae that had been placed in extraction buffer (see Material and methods). As a control, 1 μl of the raw lysate was added directly into a separate PCR reaction. (C) Whatman No.1 discs were used to purify nucleic acids from tomato plants infected with cucumber mosaic virus. The cellulose discs were added to RPA reactions with or without the presence of RT. NTCs involved adding 1 μl of water instead of DNA template. (D) Cellulose discs were used to purify nucleic acids from tomato plants that were either healthy or infected with cucumber mosaic virus and subsequently amplify them in a LAMP isothermal reaction. LAMP, loop-mediated amplification; NTC, no template control; RPA, recombinase polymerase amplification; RT, reverse transcriptase.

Fig 4. Cellulose-based paper rapidly binds DNA but releases it slowly.

(A) Whatman No.1 discs were exposed to a 1 ng/μl purified Arabidopsis genomic DNA solution for different amounts of time before washing for one minute and transferring to a PCR reaction. The band intensities achieved with cellulose discs relative to the 5 minute sample appear below each band. (B) 10 ng purified Arabidopsis genomic DNA was pipetted onto Whatman No.1 discs which were then washed in 10 ml of water with gentle agitation for different lengths of time prior to transferring the disc to a PCR reaction. The band intensities achieved with cellulose discs relative to the no wash sample appear below each band. NTC, no template control.

Fig 5. Salts enhance DNA binding to cellulose-based paper.

(A) The zeta potential of Whatman No.1 (black triangles) and Hybond N (open circles) was measured across a range of pHs. (B) Whatman No.1 discs were incubated in purified Arabidopsis genomic DNA (1 ng/μl) dissolved in water or in 150 mM NaCl. DNA solution was removed from discs by spinning, and the discs were added to a PCR amplification. The band intensities achieved with cellulose discs relative to the 60 minute sample in water appear below each band.

Fig 6. Overview of the dipstick based nucleic acid purification.

(A) The cellulose dipstick consists of a 2x40 mm wax impregnated handle and a 2x4 mm nucleic acid binding zone. (B) An overview of the dipstick-based purification method in which tissue is homogenised by shaking it in a tube containing ball bearings and an appropriate extraction buffer. The dipstick is used to bind the nucleic acids by dipping it three times into the homogenate, washed by dipping it three times into a wash buffer, and eluted by dipping it three times in the amplification reaction mix. (C) Nucleic acids were purified using the cellulose dipstick method from Arabidopsis leaves infected with F. oxysporum f.sp. conglutinans (upper image) or P. syringae (lower image) and eluted into PCR reaction mixes containing pathogen-specific primers. (D) Nucleic acids were purified from tomato leaves infected with Cucumber mosaic virus using the cellulose dipstick method. The purified DNA was eluted directly into PCR amplification reaction mixes with (+RT) or without (-RT) AMV reverse transcriptase. NTCs involved adding 1 μl of water instead of using dipstick-purified nucleic acids. AMV, Avian myeloblastosis virus; NTC, no template control; RT, reverse transcriptase.

Fig 7. Cellulose dipsticks outperform a commercially available nucleic acid purification system.

(A) The time required, number of pipetting steps involved, and the costs of all consumables—including tubes and pipette tips—were calculated for purification of nucleic acids from Arabidopsis leaf tissue using either the cellulose dipstick or Agencourt AMPure paramagnetic beads. All solutions that could be prepared in advance, including lysis and wash buffers, were made and prealiquoted. The time and pipetting involved in the preparation of these solutions was not added to the tallies in the table. (B) Purified Arabidopsis DNA at different concentrations was a captured, washed, and eluted using either the cellulose dipsticks or AMPure paramagnetic beads (Beckman Coulter). The eluted DNA was used in a PCR reaction with using primers designed for the G-protein gamma subunit 1 gene. The band intensities relative to the 1 ng/μl paramagnetic bead sample appear below each band. (C) Different volumes of an Arabidopsis leaf extract were captured, washed, and eluted using either the cellulose dipsticks or AMPure paramagnetic beads and subsequently amplified in a PCR reaction as described above. The band intensities relative to the 50 μl paramagnetic bead sample appear below each band. n.a., no amplification; USD, United States Dollar.