XII Reunião Regional Nordeste SBBq
01 a 03 de dezembro de 2014 – Natal - RN
Study of the Effect of Temperature on Stability of plasmids used in
Biotechnology
Monte, J.F.S.1; Oliveira, R.M.1,2; Rocha, H.A.O.1; Silva, M. S.1,3
1
Universidade Federal do Rio Grande do Norte, Dep. de Bioquímica, RN, Brazil;
Instituto Federal de Educação, Ciência e Tecnologia do Rio Grande do Norte, RN,
Brazil; 3Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa,
Lisboa, Portugal
2
INTRODUCTION. Plasmids (pDNA) are extrachromosomal DNA molecules double
strand that contain genetic information and have the ability to replicate
autonomously, independently of chromosomal DNA. Moreover, they have the
peculiarity of being covalently closed, presenting itself in the circular shape that can
be altered by various environmental and / or host factors and thus exhibit different
isoforms, as supercoiled, circular open, linear, denatured, that interfere with the
stability of the pDNA. Currently, the pDNA are associated with two conflicting aspects
of therapeutic interest, drug resistance and treatment based on DNA, such as gene
therapy and DNA vaccines. OBJECTIVE. The aim of this study was to evaluate the
stability of plasmids pVAX1, pVAX1LacZ and MSPpVAX1 (a plasmid encoding MSP
– major surface protease from Trypanosoma brucei parasite), when subjected to
different temperatures. MATERIAL AND METHODS. E. coli DH5-alpha cells were
transformed with the plasmids to be tested and submitted to different temperatures,
such as -80°C, -20°C, 4°C, room temperature, 37°C and 42°C and then isoforms
were analyzed through electrophoresis on a 1% agarose gel. RESULTS AND
DISCUSSION. The results of this study showed that higher temperatures leads to the
degradation of pDNA, thereby changing its original conformation (supercoiled) and
becomes relaxed. In addition, the transformation efficiency was also affected,
decreasing with increasing temperature. CONCLUSION. These results show that
there is an effect of temperature on the stability of plasmids, because the higher the
temperature, more relaxed the pDNA conformation, and for use in DNA vaccines and
gene therapy, this effect is not desirable, since they lose their stability, in addition it is
necessary that pDNA is in its supercoiled conformation for great transfection
efficiency, so it is important that the plasmids used in biotechnology are stored at low
temperatures for a better use of the pDNA.
Key words: plasmid, temperature, gene therapy, DNA vaccines
Support: CNPq and CAPES