Foram analisadas 80 amostras de casos forenses criminais do Laboratório de DNA do Instituto de Criminalística de São Paulo. O DNA foi extraído de materiais cadavéricos, vestígios de crimes sexuais e de locais de crimes. A quantificação se deu por PCR em tempo real e a amplificação por PCR, em 2 reações triplex, dos miniSTRs non-CODIS D10S1248, D14S1434 e D22S1045 (NCO01) e D1S1677, D2S441 e D4S2364 (NCO02), com posterior eletroforese capilar para detecção dos produtos fluorescentes. Avaliou-se o grau de conservação das amostras e a qualidade dos perfis genéticos obtidos. Houve concordância nas dificuldades entre diferentes amostras quando comparado com as abordagens tradicionais. Confirmou-se a sensibilidade e robustez dos sistemas utilizados, bem como a vantagem em se aumentar o número de regiões analisadas, principalmente em casos complexos, mas foram constatadas desvantagens de procedimentos tipo in-house. É necessário o estudo das frequências alélicas destes loci para a população brasileira, que possam ser incorporadas nos cálculos estatísticos dos laudos. / 80 forensic casework samples from the DNA Laboratory of the Criminalistics Institute of São Paulo were analyzed. DNA was extracted from corpse, sexual assault and crime scene evidence. The extractions were quantified using real-time PCR, amplified by two triplex PCR reactions of the non-CODIS miniSTRs D10S1248, D14S1434, D22S1045 (NC01) and D1S1677, D2S441, D4S2364 (NC02), followed by capillary electrophoresis to detect the fluorescent products. The sample preservation and quality of the genetic profiles obtained were evaluated. The results were compared to the previous obtained with the routine techniques employed in the laboratory and there was concordance in the relative difficulties. The sensibility and robustness of the systems employed were confirmed, as well as the advantages in increasing the number of loci studied, mainly in complex cases, although the in-house procedures were considered a disadvantage. The establishment of brazilian population allele frequencies for these loci is necessary for the statistical calculations of the forensic reports.

Foram analisadas 80 amostras de casos forenses criminais do Laboratório de DNA do Instituto de Criminalística de São Paulo. O DNA foi extraído de materiais cadavéricos, vestígios de crimes sexuais e de locais de crimes. A quantificação se deu por PCR em tempo real e a amplificação por PCR, em 2 reações triplex, dos miniSTRs non-CODIS D10S1248, D14S1434 e D22S1045 (NCO01) e D1S1677, D2S441 e D4S2364 (NCO02), com posterior eletroforese capilar para detecção dos produtos fluorescentes. Avaliou-se o grau de conservação das amostras e a qualidade dos perfis genéticos obtidos. Houve concordância nas dificuldades entre diferentes amostras quando comparado com as abordagens tradicionais. Confirmou-se a sensibilidade e robustez dos sistemas utilizados, bem como a vantagem em se aumentar o número de regiões analisadas, principalmente em casos complexos, mas foram constatadas desvantagens de procedimentos tipo in-house. É necessário o estudo das frequências alélicas destes loci para a população brasileira, que possam ser incorporadas nos cálculos estatísticos dos laudos. / 80 forensic casework samples from the DNA Laboratory of the Criminalistics Institute of São Paulo were analyzed. DNA was extracted from corpse, sexual assault and crime scene evidence. The extractions were quantified using real-time PCR, amplified by two triplex PCR reactions of the non-CODIS miniSTRs D10S1248, D14S1434, D22S1045 (NC01) and D1S1677, D2S441, D4S2364 (NC02), followed by capillary electrophoresis to detect the fluorescent products. The sample preservation and quality of the genetic profiles obtained were evaluated. The results were compared to the previous obtained with the routine techniques employed in the laboratory and there was concordance in the relative difficulties. The sensibility and robustness of the systems employed were confirmed, as well as the advantages in increasing the number of loci studied, mainly in complex cases, although the in-house procedures were considered a disadvantage. The establishment of brazilian population allele frequencies for these loci is necessary for the statistical calculations of the forensic reports.

The use of reporter gene fusions to assess cellular processes such as protein targeting and regulation of transcription or translation is established technology in archaeal, bacterial, and eukaryal genetics. Fluorescent proteins or enzymes resulting in chromogenic substrate turnover, like β-galactosidase, have been particularly useful for microscopic and screening purposes. However, application of such methodology is of limited use for strictly anaerobic organisms due to the requirement of molecular oxygen for chromophore formation or color development. We have developed β-lactamase from Escherichia coli (encoded by bla) in conjunction with the chromogenic substrate nitrocefin into a reporter system usable under anaerobic conditions for the methanogenic archaeon Methanosarcina acetivorans. By using a signal peptide of a putative flagellin from M. acetivorans and different catabolic promoters, we could demonstrate growth substrate-dependent secretion of β-lactamase, facilitating its use in colony screening on agar plates. Furthermore, a series of fusions comprised of a constitutive promoter and sequences encoding variants of the synthetic tetracycline-responsive riboswitch (tc-RS) was created to characterize its influence on translation initiation in M. acetivorans. One tc-RS variant resulted in more than 11-fold tetracycline-dependent regulation of bla expression, which is in the range of regulation by naturally occurring riboswitches. Thus, tc-RS fusions represent the first solely cis-active, that is, factor-independent system for controlled gene expression in Archaea.

RNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3′ processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. How Pol II elongation rates are influenced by splicing is not well understood. We generated a family of inducible gene constructs containing increasing numbers of introns and exons, which were stably integrated in human cells to serve as actively transcribing gene loci. By monitoring the association of the transcription and splicing machineries on these genes in vivo, we showed that only U1 snRNP localized to the intronless gene, consistent with a splicing-independent role for U1 snRNP in transcription. In contrast, all snRNPs accumulated on intron-containing genes, and increasing the number of introns increased the amount of spliceosome components recruited. This indicates that nascent RNA can assemble multiple spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo, Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors showed that polymerase elongation rates were uncoupled from ongoing splicing. This study shows that transcription elongation kinetics proceed independently of splicing at the model genes studied here. Surprisingly, retention of polyadenylated mRNA was detected at the transcription site after transcription termination. This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end.