DNA Sequencing: Methods, Strategies and Protocols in molecular biology research horizons

DNA sequencing approaches in the analysis of small RNA transcripts, and duplicated genes coding for enzyme inhibitors in wild and cultivated Solanum species. In recent years the exploitation of sequenced genomes has made possible the deepening of knowledge on how many genes are contained in the genomes of higher organisms. The identification of thousands of functional RNAs showed that sequenced genomes contain much more genes than previously sought. We cloned a library of RNAs sized 60-500 bases mouse and identified thirty small RNAs isolated from the developing embryo brain, the major number of them belonging to H/ACA and C/D box snoRNAs. Many of these sRNAs and snoRNAs are coded in introns of protein coding and nonprotein coding transcripts. The small RNAs can form secondary structures with free energy ranging from -3.4 to -70 kcal/mol. Three-dimensional architectural motifs are increasingly recognized as determinants of RNA functionality. Such motifs can encode spatial information required for interaction with biomolecules. Localisation on the genome databases using the UCLA Santa Cruz server showed a high conservation in these short sequences with overlapping regions of other genomes. Most of these new short RNAs have been identified today with an ENSEMBL identification number, but in our sequences there are 5' or 3' ends differences, probably relative to processing events and enzyme modifications. In the Phylogenetic analysis of Kunitz-type protease inhibitors in Slananceae, we showed a new clustering of miraculin sequences and group C inhibitors.