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.
Highlights
The ability to amplify and detect specific DNA sequences is a powerful tool routinely used for a wide variety of applications including disease diagnostics, qualitative trait loci (QTL) selection and mutant screening
We found that a number of compounds, including chitosan and polyethylenimine (PEI), showed a strong ability to bind nucleic acids (S1A Fig) and were further tested for their ability to capture genomic DNA that could be directly amplified from the modified cellulose in a PCR reaction
The ability of cellulose-based paper to entrap or adsorb DNA under specific conditions has been extensively reported, but its use has been limited to storage or transport and not for nucleic acid purification purposes under nonprecipitating conditions [10,11,23,24,25]
Summary
The ability to amplify and detect specific DNA sequences is a powerful tool routinely used for a wide variety of applications including disease diagnostics, qualitative trait loci (QTL) selection and mutant screening. Available paramagnetic beads with a variety of different functionalised surface chemistries designed to capture and purify nucleic acids have become available, removing the need for centrifugation [9,10,11]. In these systems, a magnet is used to attract and hold the paramagnetic beads to the side of the tube to allow supernatant removal during the wash and elution steps. Even though paramagnetic particle-based nucleic acid purification is relatively fast (approximately 10 minutes) and does not require electrical equipment, it is still too complicated for applications that are performed outside the modern laboratory environment such as fieldbased point-of-need (PON) assays
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