Table of Contents | Genet. Mol. Res. 2023 (4)
Next-generation sequencing (NGS) platforms are now implemented as routine analysis for K-RAS mutation in colon cancer patients before therapy. The DNA used in NGS platforms is extracted from colon cancer formalin-fixed paraffin-embedded (FFPE) blocks. In this study, we utilized 20 FFPE colon cancer blocks. In general, a good quality DNA sample includes compact high molecular weight DNA. The quality of the extracted DNA is checked by agarose gel electrophoresis. Some samples are found to be highly degraded due to natural mechanisms like autolysis and spontaneous depurination, or bacterial contamination and extracted DNA is then routinely fragmented by sonication. In this study, PCR was performed to reconstruct larger DNA fragments rather than to amplify DNA fragments. Primerless PCR relies on the natural power of two segments of the PCR cycle to reconstruct fragmented PCR by: the capability of denatured DNA to anneal randomly to its complementary sequence (annealing), and Taq polymerase to extend the DNA at the 3’ end (extension). By repeating for 150 cycles a larger DNA fragment is generated instead of amplifying the DNA. Fragmented DNA was reconstructed by primerless PCR for 150 cycles. However, 1U of Taq polymerase was added to the PCR reaction every 50 cycles. The samples selected for this study were highly degraded. The degree of degradation of samples was visualized by running the samples on a 1% agarose gel. After running primerless PCR for 50, 100, and 150 cycles, larger fragments of highly intact DNA appeared as sharp, compact bands whereas degraded DNA was a diffuse smear. In conclusion, we have demonstrated that primerless PCR can authentically generate DNA fragments that are larger than the initial template, and the DNA polymerization of self-primed DNA fragments can increase the likelihood of successful regeneration ostensibly by reconstructing the template. Primerless PCR can help in regenerating larger DNA fragments from highly deteriorated samples, such as forensic, and ancient samples.
Essential oils (EO) are substances used by pharmaceutical, cosmetic and food industries. These compounds have useful properties for human health and well-being. Among the species cultivated for production of these substances are those of the genus Cymbopogon. Several species, subspecies, varieties, and sub-varieties are used worldwide, and studies have been conducted to elucidate the chemical features of their EO, mainly for C. citratus and C. winterianus. However, there are still species with great potential that have not yet been fully chemically characterized. In addition, EO composition is influenced by genetic and environmental factors. Along this line, we examined the chemical profile of EO of four species of the genus Cymbopogon (C. citratus, C. distans, C. flexuosus and C. winterianus) grown in southern Brazil. The EO was obtained from fresh leaf tissues by hydrodistillation. Citral was identified as the main component of C. citratus, C. flexuosus, and C. distans, whereas citronellal, citronellol, cis-geraniol and elemol were predominant in C. winterianus. The absence of a large range of compounds was noted in C. distans, which may be useful for the semi-exclusive synthesis of citral and other compounds of interest.
X-chromosome inactivation (XCI), an essential epigenetic event in female embryos, is involved in embryonic development and requires refined control mechanisms. Although studies in cattle have been contributing to the understanding of the dynamic mechanisms of XCI, no research has yet investigated XCI in elongated bovine embryos. We used qPCR to characterize the mRNA levels of five target genes (XIST, JPX, H2AFY, H2AFY2, and EZH2) related to XCI in bovine embryos at stages: 8-16-cells (72 embryos), morula (72 embryos), blastocyst (64 embryos), hatched blastocyst (64 embryos), D11 blastocyst (20 embryos), and D14 (biopsies of 4 biopsies of embryos). Our results showed the same mRNA levels of XIST, JPX, and H2AFY2 at the morula stage, which were higher than those of the other genes. However, JPX declined after the morula stage, whereas XIST and H2AFY2 maintained higher mRNA levels in BL and HB. The expression of XIST and H2AFY2 reached the lowest levels at D11 and D14. We suggest that the changes in mRNA levels of these genes in the initial stages of development are related to the onset of XCI in cattle. This study is relevant to improve our understanding of the dynamic mechanisms that act in XCI in cattle until the elongated embryo stage.