Sistema para análise da polimerização de ácidos nucleicos na reação em cadeia da polimerase

Abstract

In recent years, molecular biology has undergone major advances, being the polymerase chain reaction (PCR) one of them. In this technique, several copies of the desired fragment are obtained from a small amount of deoxyribonucleic acid (DNA). Real-time PCR, a variant of conventional PCR, is a technique used as a quantitative molecular diagnosis method in medical, forensic and biotechnological research, beocause of its speed, sensitivity, specificity and reproducibility. This work aims to design a system to evaluate, in real time, the polymerization of a specific DNA molecule using a real time PCR thermocycler. In our model, we used a 2.040 base pair fragment (cytosine-guanine content of 52%) that was generated by PCR using the plasmid pCR-bicolor as template. The amplified fragment has a T7 universal sequence at one end and a universal sequence M13 at the other. Thus, after reamplification of the PCR fragment, a 6-carboxy-fluorescein (FAM) at the T7 end and a biotin molecule at the M13 end were added. Subsequently, the biotinylated DNA was ligated on streptavidin-coated magnetic beads and the double strand was denatured in alkaline solution, keeping one strand attached to particle and another in the alkaline solution. Both strands were separated and subjected to elongation analysis of the complementary strand. Initially, the results obtained from the beads and the neutralized strand, were unsatisfactory for the planned system. The magnetic beads precipitate in the reaction, decreasing the availability of the reagents for the amplification of the complementary strand. Another important factor is that the binding of the DNA strand to the beads dissolves with the temperature of the elongation process, which makes it impossible to use the same bead for a new process. In the neutralized strand was possible to analyze the polymerization, but the sodium acetate produced in the neutralization is an inhibitor of the reaction catalytic enzyme. In addition, there is a low amount of single strand material obtained because the efficiency of the beads is inversely proportional to the size of the purified fragment. It was concluded that the system tested to date is not feasible with the use of the beads, and with the neutralized strand requires the need for further studies. An alternative to render the process feasible could be to obtai simple strand material from DNA extracted from bacteriophage M13 or a purification of the biotinylated DNA of the magnetic beads.