RESUMO
Fluorescent in situ hybridization (FISH) is a powerful method for the targeted visualization of nucleic acids in their native contexts. Recent technological advances have leveraged computationally designed oligonucleotide (oligo) probes to interrogate > 100 distinct targets in the same sample, pushing the boundaries of FISH-based assays. However, even in the most highly multiplexed experiments, repetitive DNA regions are typically not included as targets, as the computational design of specific probes against such regions presents significant technical challenges. Consequently, many open questions remain about the organization and function of highly repetitive sequences. Here, we introduce Tigerfish, a software tool for the genome-scale design of oligo probes against repetitive DNA intervals. We showcase Tigerfish by designing a panel of 24 interval-specific repeat probes specific to each of the 24 human chromosomes and imaging this panel on metaphase spreads and in interphase nuclei. Tigerfish extends the powerful toolkit of oligo-based FISH to highly repetitive DNA.
Assuntos
DNA , Sequências Repetitivas de Ácido Nucleico , Humanos , Hibridização in Situ Fluorescente/métodos , DNA/genética , Sequências Repetitivas de Ácido Nucleico/genética , Sondas de Oligonucleotídeos/genética , Sondas de DNA/genética , Oligonucleotídeos/genéticaRESUMO
BACKGROUND: Tea polysaccharide conjugate (TPC) is a naturally occurring active substance that is extracted from tea. Owing to its benefits in enhancing human immunity and antioxidant effects, TPC is widely used in culinary products. The binding mode of polysaccharides and proteins in TPC, however, has not been well studied; it may be closely related to their functional properties, especially emulsification. RESULTS: The molecular weights and monosaccharide compositions of TPC were determined by ion chromatography and high-performance gel permeation chromatography. Although the functional groups of polysaccharides and proteins were confirmed by infrared spectroscopy, the presence of proteins could not be detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and ultraviolet spectroscopy. It was hypothesized that the hydrophobic groups of the proteins in TPC were wrapped by polysaccharide chains, thus making the proteins undetectable. The rheology and interfacial protein adsorption results show that TPC forms a viscoelastic film at the oil-water interface to prevent the aggregation of oil droplets, thereby enhancing the stability of the emulsion. Based on these structural and emulsifying properties of TPC, the binding mode of polysaccharides and proteins along with their phase behavior at the oil-water interface of the emulsion was speculated. CONCLUSION: In TPC, the hydrophilic groups of the proteins are linked to polysaccharides by covalent interactions, where the hydrophobic groups are wrapped with the polysaccharide chains with the help of hydrophobic forces to form a hydrophobic core. The unique binding of polysaccharides and proteins in TPC enhances its amphiphilic properties, which can be effectively distributed at the oil-water interface and form stable emulsions. © 2023 Society of Chemical Industry.
Assuntos
Polissacarídeos , Chá , Humanos , Emulsões/química , Polissacarídeos/química , Adsorção , Chá/química , Água/químicaRESUMO
Fluorescent in situ hybridization (FISH) is a powerful method for the targeted visualization of nucleic acids in their native contexts. Recent technological advances have leveraged computationally designed oligonucleotide (oligo) probes to interrogate >100 distinct targets in the same sample, pushing the boundaries of FISH-based assays. However, even in the most highly multiplexed experiments, repetitive DNA regions are typically not included as targets, as the computational design of specific probes against such regions presents significant technical challenges. Consequently, many open questions remain about the organization and function of highly repetitive sequences. Here, we introduce Tigerfish, a software tool for the genome-scale design of oligo probes against repetitive DNA intervals. We showcase Tigerfish by designing a panel of 24 interval-specific repeat probes specific to each of the 24 human chromosomes and imaging this panel on metaphase spreads and in interphase nuclei. Tigerfish extends the powerful toolkit of oligo-based FISH to highly repetitive DNA.
