Extração e amplificação de DNA de bactérias em Dispositivos microfluídicos descartáveis de poliéstertoner (PT)

Abstract

The microfluidic enables the manipulation of small volumes of fluids in micrometer channels providing several analytical advantages and applications in different areas. The evolution of microscale analysis seeks to revolutionize the analytical techniques and the conventional laboratory manipulations. In this work, polyester-toner (PT) microfluidic devices were constructed to perform dynamic solid phase DNA extraction (dSPE) with magnetic silica beads and amplification by Polymerase Chain Reaction (PCR) from Azospirillum brasilense AbV5 and Escherichia coli BL21 transformed with pGEX-4T-3 plasmid. By the eluted fractions quantification in the DNA extraction were elaborated the graphics of the elution profile for evaluation of the amount of DNA extracted before and after the magnetic silica beads agitation and the calculation of extraction efficiency. The DNA amplification by PCR was performed on a homemade system by heating mediated by infrared radiation (IR) and temperature controlled by a thermocouple with the aid of LabVIEW software. The DNA extraction performed in PT microchips showed to be fast, with high efficient extraction and purity, that can be amplified in downstream PCR amplification. Amplified products were obtained by PCR using the primer pair Y1 and Y3 correspond to the 16S rDNA gene of the DNA of A. brasilense AbV5 bacteria and the primer pair blaTEM forward and blaTEM reverse correspond to the antibiotic resistance encoding gene of the DNA of E. coli BL21 bacteria. The amplified products were separated by electrophoresis on Bioanalyzer showing fluorescence peaks corresponding to 1500 and 1150 base pairs for the A. brasilense AbV5 and E. coli BL21 bacteria, respectively. The results demonstrate for the first time the successful use of PT microchips for DNA analysis from bacteria.