Banca de DEFESA: ALLAN ROBERTO DIAS NUNES

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
DISCENTE : ALLAN ROBERTO DIAS NUNES
DATA : 28/02/2017
HORA: 08:30
LOCAL: Sala SS1
TÍTULO:

Oligonucleotides: design, production and application for detection flavivirus

PALAVRAS-CHAVES:

Oligodeoxyribonucleotide, enzymatic synthesis, primer design, flavivirus

PÁGINAS: 108
GRANDE ÁREA: Ciências Biológicas
ÁREA: Bioquímica
RESUMO:

Oligonucleotides are small nucleic acid molecules with great biotechnological importance in various fields of biology and medicine. The genetic identification of pathogens through the PCR technique and its variations is among its main applications. The design and production of oligonucleotides are critical points for their application. The most commonly used technique for the production of DNA oligonucleotides is chemical synthesis. Despite its effectiveness, this technique cannot be performed in-house, making the user very dependent on a supplier. This work aimed to design new oligonucleotides with potential for different applications, to develop a new method to produce them and to validate their application in a PCR protocol for flavivirus detection. Initially, a set of universal primers for identification of flavivirus was developed. In the first stage of the work, 1442 complete genomes of different representatives of the genus flavivirus were aligned for selection of conserved regions (CRs). 26 CRs were selected, which allowed the design of 66 universal primers. The 10 primers best classified according to their size, Tm and degeneration, are located in the NS5 protein and were chosen for the validation of the PCR protocol. In parallel, specific primers that generate fragments of different sizes for the Zika virus and the Dengue virus serotypes were designed. Thus, a nested-PCR RT system was developed which, in the first step of amplification, generates a fragment ranging from 800-806 bp, allowing the identification of any flavivirus by means of amplicon sequencing. In the second step, fragments of different sizes can differentiate zika virus and the four dengue serotypes in agarose gel. In the second stage of the work, an enzymatic method for the production of DNA oligonucleotides was developed. The proposed method is based on rolling circle replication (RCA) and comprises four enzymatic steps: (1) phosphorylation of the 5 'end of the target sequence, (2) circularization of the target sequence, (3) polymerization of a new single strand DNA containing the oligonucleotides of interest and, finally, (4) a restriction assay to release the oligonucleotides. All steps were performed in a single tube, adding the enzymes with their respective buffer solutions. The fragments generated were separated using 8% polyacrylamide gel electrophoresis (PAGE) and visualized by silver staining. Potentially, any oligonucleotide that has no degenerate bases can be produced by the proposed enzymatic method. To illustrate, the production of three variations of aptamer 31-TBA, a single-stranded oligonucleotide that has anticoagulant action, was presented. The universal oligonucleotides for flavivirus detection could not be synthesized by this enzymatic method because it has some degenerate bases in its composition, but some constructs for the production of primers for detection of ZIKV were developed.

MEMBROS DA BANCA:
Externo à Instituição - ANA ISABELA LOPES SALES - UnP
Presidente - 1880243 - DANIEL CARLOS FERREIRA LANZA
Interno - 1513597 - JOAO PAULO MATOS SANTOS LIMA