RESUMEN
Herein we present TmPrime, a computer program to design oligonucleotide sets for gene assembly by both ligase chain reaction (LCR) and polymerase chain reaction (PCR). TmPrime offers much flexibility with no constraints on the gene and oligonucleotide lengths. The program divides the long input DNA sequence based on the input desired melting temperature, and dynamically optimizes the length of oligonucleotides to achieve homologous melting temperatures. The output reports the melting temperatures, oligonucleotide sequences and potential formation of secondary structures. Our program also provides functions on sequence pooling to separate long genes into smaller pieces for multi-pool assembly and codon optimization for expression. The software has been successfully used in the design and synthesis of green fluorescent protein fragment (GFPuv) (760 bp), human protein kinase B-2 (PKB2) (1446 bp) and the promoter of human calcium-binding protein A4 (S100A4) (752 bp) using real-time PCR assembly with LCGreen I, which offers a novel approach to compare the efficiency of gene synthesis. The purity of assembled products is successfully estimated with the use of melting curve analysis, which would potentially eliminate the necessity for agarose gel electrophoresis. This program is freely available at http://prime.ibn.a-star.edu.sg.
Asunto(s)
Genes Sintéticos , Oligonucleótidos/química , Programas Informáticos , Humanos , Reacción en Cadena de la Ligasa , Reacción en Cadena de la Polimerasa , Proteínas/genética , Análisis de Secuencia de ADNRESUMEN
Here we present a simple, highly efficient, universal automatic kinetics switch (AKS) gene synthesis method that enables synthesis of DNA up to 1.6kbp from 1nM oligonucleotide with just one polymerase chain reaction (PCR) process. This method eliminates the interference between the PCR assembly and amplification in one-step gene synthesis and simultaneously maximizes the amplification of emerged desired DNA by using a pair of flanked primers. In addition, we describe an analytical model of PCR gene synthesis based on the thermodynamics and kinetics of DNA hybridization. The kinetics difference between standard PCR amplification and one-step PCR gene synthesis is analyzed using this model and is validated using real-time gene synthesis with eight gene segments (318-1656bp). The effects of oligonucleotide concentration, stringency of annealing temperature, annealing time, extension time, and PCR buffer conditions are examined systematically. Analysis of the experimental results leads to new insights into the gene synthesis process and aids in optimizing gene synthesis conditions. We further extend this method for multiplexing gene assembly with a total DNA length up to 5.74kbp from 1nM oligonucleotide.
Asunto(s)
Genes/genética , Reacción en Cadena de la Polimerasa/métodos , Automatización , Tampones (Química) , Cartilla de ADN/genética , Humanos , Cinética , Oligodesoxirribonucleótidos/genética , TemperaturaRESUMEN
We have recently described the MELC/TIS fluorescence robot technology that is capable of colocalizing at least a hundred different molecular cell components in one cell. The technology reveals new hierarchical properties of protein network organisation, referred to as the toponome, in which topologically confined protein clusters are interlocked within the structural framework of the cell. In this study we have applied MELC/TIS to construct a three-dimensional toponome map of the cell nucleus of a single human hepatocyte undergoing apoptosis. The map reveals six different spatially separated toponome domains in the nuclear interior of one apoptotic cell. In the drive to decipher the apoptosis-specific molecular network on the single cell level, the present toponome map is a first milestone towards the construction of much larger maps addressing hundreds of molecular cell components across the stages of apoptosis.
Asunto(s)
Apoptosis/fisiología , Núcleo Celular , Hepatocitos , Imagenología Tridimensional/métodos , Núcleo Celular/química , Núcleo Celular/metabolismo , Células Cultivadas , Hepatocitos/citología , Hepatocitos/fisiología , Humanos , Microscopía Fluorescente/métodosRESUMEN
Temporal and spatial regulation of proteins contributes to function. We describe a multidimensional microscopic robot technology for high-throughput protein colocalization studies that runs cycles of fluorescence tagging, imaging and bleaching in situ. This technology combines three advances: a fluorescence technique capable of mapping hundreds of different proteins in one tissue section or cell sample; a method selecting the most prominent combinatorial molecular patterns by representing the data as binary vectors; and a system for imaging the distribution of these protein clusters in a so-called toponome map. By analyzing many cell and tissue types, we show that this approach reveals rules of hierarchical protein network organization, in which the frequency distribution of different protein clusters obeys Zipf's law, and state-specific lead proteins appear to control protein network topology and function. The technology may facilitate the development of diagnostics and targeted therapies.
Asunto(s)
Microscopía Fluorescente/métodos , Proteínas/análisis , Proteínas/metabolismo , Dermatitis Atópica/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Espectrometría de Masas , Patología/métodos , Proteómica/métodos , Psoriasis/metabolismo , Reproducibilidad de los Resultados , Piel/metabolismoRESUMEN
We have correlated transcriptomics, proteomics and toponomics analyses of hippocampus tissue of inbred C57BL/6 mice to analyse the interrelationship of expressed genes and proteins at different levels of organization. We find that transcriptome and proteome levels of function as well as the topological organization of synaptic protein clusters, detected by toponomics at physiological sites of hippocampus CA3 region, are all largely conserved between different mice. While the number of different synaptic states, characterized by distinct synaptic protein clusters, is enormous (>155,000), these states together form synaptic networks defining distinct and mutually exclusive territories in the hippocampus tissue. The findings provide insight in the systems biology of gene expression on transcriptome, proteome and toponome levels of function in the same brain subregion. The approach will lay the ground for designing studies of neurodegeneration in mouse models and human brains.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Hipocampo/metabolismo , Proteómica/métodos , Animales , Electroforesis en Gel Bidimensional , Hipocampo/anatomía & histología , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía FluorescenteRESUMEN
The fluorescence robot imaging technology multi-epitope-ligand-cartography/toponome imaging system has revolutionized the field of proteomics/functional genomics, because it enables the investigator to locate and decipher functional protein networks, the toponome, consisting of hundreds of different proteins in a single cell or tissue section. The technology has been proven to solve key problems in biology and therapy research. It has uncovered a new cellular transdifferentiation mechanism of vascular cells giving rise to myogenic cells in situ and in vivo; a finding that has led to efficient cell therapy models of muscle disorders, and discovered a new target protein in sporadic amyotrophic lateral sclerosis by hierarchical protein network analysis, a finding that has been confirmed by a mouse knockout model. A lead target protein in tumor cells that controls cell polarization as a mechanism that is fundamental for migration and metastasis formation has also been uncovered, and new functional territories in the CNS defined by high-dimensional synaptic protein clusters have been unveiled. The technology can be effectively interlocked with genomics and proteomics to optimize time-to-market and the overall attrition rate of new drugs. This review outlines major proofs of principle with an emphasis on neurotoponomics.
Asunto(s)
Proteínas del Tejido Nervioso/análisis , Orgánulos/química , Proteómica/métodos , Biología de Sistemas , Animales , HumanosRESUMEN
This chapter presents TmPrime, a computer program to design oligonucleotide for both ligase chain reaction (LCR)- and polymerase chain reaction (PCR)-based de novo gene synthesis. The program divides a long input DNA sequence based on user-specified melting temperatures and assembly conditions, and dynamically optimizes the length of oligonucleotides to achieve homologous melting temperatures. The output reports the melting temperatures, oligonucleotide sequences, and potential formation of secondary structures in a PDF file, which will be sent to the user via e-mail. The program also provides functions on sequence pooling to separate long genes into smaller pieces for multipool assembly and codon optimization for expression based on the highest organism-specific codon frequency. This software has been successfully used in the design and synthesis of various genes with total length >20 kbp. This program is freely available at http://prime.ibn.a-star.edu.sg.
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Oligodesoxirribonucleótidos/biosíntesis , Oligodesoxirribonucleótidos/genética , Programas Informáticos , Secuencia de Bases , Codón/genética , Biología Computacional , Humanos , Reacción en Cadena de la Ligasa , Datos de Secuencia Molecular , Desnaturalización de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Reacción en Cadena de la PolimerasaRESUMEN
With an aim to develop peptide-based protein capture agents that can replace antibodies for in vitro diagnosis, an ultra-high-throughput screening strategy has been investigated by automating labor-intensive, time-consuming processes that are the construction of peptide libraries, sorting of positive beads, and peptide sequencing through analysis of tandem mass spectrometry data. Although instruments for automation, such as peptide synthesizers and automatic bead sorters, have been used in some groups, the overall process has not been well optimized to minimize time, cost, and efforts, as well as to maximize product quality and performance. Herein we suggest and explore several solutions to the existing problems with the automation of the key processes. The overall process optimization has been done successfully in orchestration with the technologies such as rapid cleavage of peptides from beads and semiautomatic peptide sequencing that we have developed previously. This optimization allowed one-round screening, from peptide library construction to peptide sequencing, to be completed within 4 to 5 days. We also successfully identified a 6-mer ligand for carcinoembryonic antigen-cell adhesion molecule 5 (CEACAM 5) through three-round screenings, including one-round screening of a focused library.
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Automatización de Laboratorios , Ensayos Analíticos de Alto Rendimiento , Fragmentos de Péptidos/aislamiento & purificación , Biblioteca de Péptidos , Biomarcadores de Tumor/agonistas , Biomarcadores de Tumor/química , Antígeno Carcinoembrionario/química , Técnicas Químicas Combinatorias/instrumentación , Técnicas Químicas Combinatorias/métodos , Diagnóstico , Proteínas Ligadas a GPI/agonistas , Proteínas Ligadas a GPI/química , Humanos , Ligandos , Microesferas , Fragmentos de Péptidos/química , Análisis de Secuencia de Proteína/métodos , Espectrometría de Masas en TándemRESUMEN
This protocol details sample preparation and measurement procedures for a fluorescence technology capable of colocalizing hundreds of different proteins in a cell or tissue section. The procedure relies on fixation of samples and on the use of dye-conjugated tag libraries. To colocalize proteins, a sample is placed on the microscope stage of an imaging system (toponome imaging system (TIS)) performing sequential cycles of tag-dye incubation, imaging and bleaching to generate images for each localization cycle. TIS overcomes the spectral limitations of traditional fluorescence microscopy. Image processing reveals toponome maps, uncovering the coexistence of proteins at a location (protein clusters). The approach provides direct insight into the topological organization of proteins on a proteomic scale for the first time. If, for example, two dyes are used per cycle, 18 proteins in 4 visual fields can be colocalized in 21 h. Parallel TIS procedures using more than two dyes per cycle enhance the throughput.