mRNA genotyping by gold nanoprobes

Dissertação para obtenção do Grau de Mestre em BiotecnologiaBionanotechnology has emerged as a field with great potential for molecular diagnose. Namely, the use of gold nanoparticles has allowed the development of molecular diagnostic methods with greater sensitivity and specificity at a fraction of the cost inherent to conventional techniques. The present work assessed the ability of gold nanoprobes to detect targets with single base differences in RNA molecules following the colorimetric non-cross-linking method. As proof-of-concept, gold nanoprobes were designed, synthesized and characterized to detect three different SNPs (c.2731C>T, c.3232A>G and c.3238G>A) in the BRCA1 gene, a gene associated with inherited breast cancer. Reference materials, susceptible to be used for the calibration of the method, were created by cloning genomic fragments amplified from biological samples containing the sequences of interest in an appropriate vector for subsequent in vitro/in vivo transcription. Initially, the ability of target recognition by the gold nanoprobes was assessed using synthetic oligonucleotides targets alone and spiked-in total RNA of Saccharomyces cerevisiae, and later using transcripts synthetized in vitro/in vivo. This study revealed the capacity of target detection up to 0.25% of complementary target/total RNA, for a final concentration of complementary target of 0.12pmol/μL. It was also possible to detect and discriminate both c.3232A>G SNP alleles using only 0.08pmol/μL of in vitro transcript. For the in vivo transcript samples the results were inconclusive