The study of protein function usually requires the use of a cloned version of the gene for protei... more The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single-gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator(TM) DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This tutorial is part of the...
The correct identification of all human genes, and their derived transcripts, has not yet been ac... more The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. I...
Conductometric monitoring of protein-protein and protein-sterol interactions is here proved feasi... more Conductometric monitoring of protein-protein and protein-sterol interactions is here proved feasible by coupling quartz crystal microbalance with dissipation monitoring (QCM_D) to nucleic acid programmable protein arrays (NAPPA). The conductance curves measured in NAPPA microarrays printed on quartz surface allowed the identification of binding events between the immobilized proteins and the query. NAPPA allows the immobilization on the quartz surface of a wide range of proteins and can be easily adapted to generate innumerous types of biosensors. Indeed multiple proteins on the same quartz crystal have been tested and envisaged proving the possibility of analyzing the same array for several distinct interactions. Two examples of NAPPA-based conductometer applications with clinical relevance are presented herein, the interaction between the transcription factors Jun and ATF2 and the interaction between Cytochrome P540scc and cholesterol.
The study of protein function usually requires the use of a cloned version of the gene for protei... more The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single-gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator(TM) DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This tutorial is part of the...
The correct identification of all human genes, and their derived transcripts, has not yet been ac... more The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. I...
Conductometric monitoring of protein-protein and protein-sterol interactions is here proved feasi... more Conductometric monitoring of protein-protein and protein-sterol interactions is here proved feasible by coupling quartz crystal microbalance with dissipation monitoring (QCM_D) to nucleic acid programmable protein arrays (NAPPA). The conductance curves measured in NAPPA microarrays printed on quartz surface allowed the identification of binding events between the immobilized proteins and the query. NAPPA allows the immobilization on the quartz surface of a wide range of proteins and can be easily adapted to generate innumerous types of biosensors. Indeed multiple proteins on the same quartz crystal have been tested and envisaged proving the possibility of analyzing the same array for several distinct interactions. Two examples of NAPPA-based conductometer applications with clinical relevance are presented herein, the interaction between the transcription factors Jun and ATF2 and the interaction between Cytochrome P540scc and cholesterol.
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Papers by Fernanda Festa