|1282-3||PCR PRODUCT (linear PCR amplicon) AND PLASMID STANDARDIZATION FOR THE CALIBRATION CURVE|
|Autores:||Vivian Fonseca Gonzaga (USP - Universidade de São Paulo) ; Tatiana Lopes (USP - Universidade de São Paulo) ; Daniela de Araújo Viana Marques (USP - Universidade de São Paulo) ; João Vitor Dutra Molino (USP - Universidade de São Paulo) ; Joas Lucas da Silva (USP - Universidade de São Paulo) ; Mario Hiroyuki Hirata (USP - Universidade de São Paulo) ; Priscila Gava Mazzola (UNICAMP - Universidade Estadual de Campinas) ; Maria Silvia Viccari Gatti (UNICAMP - Universidade Estadual de Campinas) ; Adalberto Pessoa Júnior (USP - Universidade de São Paulo) |
Aqueous two polymeric phase system (ATPPS) composed of polyacrylic acid and polyethylene glycol (PEG/NaPA) are proposed as an alternative for purification, separation and concentration of biomolecules and bioparticles, preventing loss of activity or desirable properties during the process. This system was used to concentrate Human Adenovirus type 5 (HAd-5). To reach the proposed goal, optimal method of standardizing the calibration curve was established. Among the various types of standard DNA, plasmid DNA, especially the uncut circular one, is the most common choice due to its high stability and reproducibility. However, the circular plasmid standard may cause some errors changing the conformational types of DNA and thus destabilizing the standard curve. Techniques using circular plasmid DNA and PCR product (linear PCR amplicon) were compared to examine the effect of DNA structural on amplification efficiency of qPCR standard curve. First, amplification of the fragments containing the virus (HAd-5) was performed using pairs of primers (sense and antisense) by polymerase chain reaction (PCR) and the virus DNA presence was confirmed through agarose gel electrophoresis. The selected fragments were inserted into the pGEM plasmid using pGEM plasmid ®-T Easy Vector Systems. Second, the PCR reaction product (linear PCR amplicon) was extracted, concentrated and stored to be use in the standard curve. DNA quantification of modified plasmid and linear PCR amplicon was obtained using NanoDrop ®.These two products were tested for four weeks to evaluate the reproducibility of each one on standardizing the calibration curve. Samples were diluted in the order of 10 (10-2 to 10-9) and analyzed by qPCR. Results showed that mean values of standard curves efficiency for both methods, PCR product (linear PCR amplicon) and using modified plasmid with target DNA sequence, were similar, around 86% without much difference. Through this study it is concluded that the standard curve can be performed using either the PCR product of virus DNA or the modified plasmid.
Palavras-chave: linear PCR amplicon, PLASMID, CALIBRATION CURVE, Adenovirus