DNA methylation patterns of steroid receptor genes ESR1, ESR2 and PGR in deep endometriosis compromising the rectum

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação da expressão gênica e de lesões no DNA de índividuos portadores de diabetes mellitus tipo 2, dislipidemia e periodontite crônica

Pós-graduação em Odontologia - FOAR

Avaliação da diluição do DNA extraido de material parafinado para amplificação em PCR

Introduction: Several reasons may lead to the failure of polymerase chain reaction (PCR) using DNA purified from paraffin-embedded materials: presence of inhibitors and degradation of target DNA. DNA dilution will often reduce the concentration of potential inhibitors and still contain enough DNA to allow PCR amplification. Objective: To evaluate the dilution influence of DNA purified from paraffin-embedded materials on β-globin PCR amplification. Material and Method: Paraffin-embedded blocks from 30 patients with oropharynx squamous cell carcinomas, diagnosed and treated at the Oral Oncology Center were selected. DNA extraction was performed using QIAmp minikit (Quiagen). DNA was quantified and evaluated for purity by spectrophotometer analysis. Two groups were formed with different amounts of DNA: group I had the originally extracted DNA and group II had the same DNA, however diluted with ultrapure water addition. PCR was performed in both groups using oligonucleotides for human β-globin gene. Results: For Group I, amplification of the β-globin gene sequence was successful in 33.33% of the samples and for Group II, in 23.33%. Conclusion: Dilution of the DNA extracted of paraffin-embedded materials did not modify statistically the amount of positive samples β-globin gene amplified in PCR, although the results suggest that this is a way to increase the method for efficacy amplification of PCR.

Comparação de métodos para extração de dna na reação em cadeia da polimerase para detecção de Salmonella enterica sorovar enteritidis em produtos avícolas

The identification of Salmonella spp. in food samples by microbiological diagnosis is time consuming, with approximately five different stages, requiring about 120 hours until the final result. The utilization of the polymerase chain reaction technique (PCR) can reduce this time, but substances present in samples may affect the reaction. The present work aimed to compare DNA extraction by thermic treatment and by the use of cetyltrimethil ammonium bromide (CTAB), in products originated from poultry houses corresponding to raw material (meat meal) and experimentally contaminated drag swabs. Materials obtained from the extractions were submitted to PCR, utilizing a pair of initiator oligonucleotides for amplification of Sdf 1 gene fragments. Comparing the methods of extraction, it was observed that when CTAB was employed, SE was detected in 70% of meat meal and in 80% of drag swabs, while the thermic treatment method yielded positive results in 20% of meat meal and in 40% of drag swabs. SE was detected under both methods utilized for DNA extraction, but the use of CTAB detected a greater number of positive samples, compared with thermal treatment.