DISPOSABLE MICROPIPPETTE-TIP FOR PURIFYING NUCLEIC ACIDS

MIRNA LORENA SANCHEZ; GIMENEZ, CLAUDIA; MARTÍNEZ, LEANDRO J.; GRASSELLI, MARIANO

Guaruja

Conferencia; Biopartitioninig and Purification Conference (BPP 2019); 2019

The use of the pipette-tip has become crucial and essential in chemical/biological fields, which deal with various micro-chemical reactions that require accurate control and transfer of tens to hundred microliters of chemical reactant solutions [1,2]. It has encouraged researchers to examine and modify the conventional pipette-tip and thus provide additional functionalities other than accurate transfer of solutions such as filtering, fractionation and pre-chemical treatment [3-7].In this work, a novel prototype based on functional micropipette-tip with an internal PVA (polyvinyl alcohol) coating with silica nanoparticles embedded stabilized by gamma radiation induced polymerization (GRIP) was prepared.It will be described the preparation, characterization and functional testing of that dispositive called DNATip, able to interact with PCR DNA fragments or plasmid DNA in order to purify them from a complex sample in a practical and simple way. To determine the optimal preparation protocol of the DNATip, pH of the coating solution and the type and concentration of the silica particles were tested. Results showed that coatings prepared with fumed silica were more homogeneous and stable over the time. In addition, adsorption capacity of plasmid DNA using fumed silica was higher in comparison with the obtained with silica particles of 40-63 μm or 20-40 nm in diameter. It was found that this material prepared at neutral pH, showed the best plasmid DNA adsorption values and the highest silica concentration allowed a greater plasmid DNA recovery.This prepared DNATip was able to purify PCR DNA-fragments of 500 pb from a sample contaminated with BSA. The purified fragments had the quality enough to be sequenced or enzymatically digested.The development of new devices to be applied to DNA purification have continuous attracted attention in order to simplify procedures, cost and improvements in product quality.Results here showed, let us propose this new disposable material for nucleic acids purification as a novel lab-on-a-tip device which one could effectively improve procedures.References: [1] DN Miller et al., Appl. Environ. Microbiol. 1999, 65, 11, p. 4715-4724. [2] H Tian, AF Hühmer, JP Landers, Anal. Biochem. 2000, 283, 2, p.175-191. [3] J Rappsilber, M Mann, Y Ishihama, Nat. Protoc. 2007, 2, 8, p. 1896. [4] D Choi et al., Nano Energy 2017, 38. [5] H Nakagami, in Plant Proteomics 2014, pp. 595-607, Humana Press, Totowa, NJ. [6] J Rappsilber, Y Ishihama, M Mann, Anal. Chem 2003, 75, 3, p. 663-670. [7] Y Ishihama, J Rappsilber, M Mann, J. Proteome Res. 2006, 5, 4, p. 988-994.Keywords: lab-on-a-tip, purification, nucleic-acids, PCR.Development Agency: El Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).