Simple and cost-effective restriction endonuclease analysis of human adenoviruses. 2014

Arun Kumar Adhikary, and Nozomu Hanaoka, and Tsuguto Fujimoto
Unit of Microbiology, Faculty of Medicine, AIMST University, Semeling, 08100 Bedong, Kedah Darul Aman, Malaysia ; Infectious Disease Surveillance Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.

Restriction endonuclease analyses (REAs) constitute the only inexpensive molecular approach capable of typing and characterizing human adenovirus (HAdV) strains based on the entire genome. However, the application of this method is limited by the need for time-consuming and labor-intensive procedures. We herein developed a simple and cost-effective REA for assessing HAdV. The method consists of (1) simple and cost-effective DNA extraction, (2) fast restriction endonuclease (RE) digestion, and (3) speedy mini agarose gel electrophoresis. In this study, DNA was isolated according to the kit-based method and 21.0 to 28.0  μg of viral DNA was extracted from prototypes (HAdV-1, HAdV-3, HAdV-4, and HAdV-37) in each flask. The amount of DNA ranged from 11.4 to 57.0  μg among the HAdV-3 (n=73) isolates. The obtained viral DNA was found to be applicable to more than 10 types of REAs. Fast-cut restriction endonucleases (REs) were able to digest the DNA within 15 minutes, and restriction fragments were easily separated via horizontal mini agarose gel electrophoresis. The whole procedure for 10 samples can be completed within approximately six hours (the conventional method requires at least two days). These results show that our REA is potentially applicable in many laboratories in which HAdVs are isolated.

UI MeSH Term Description Entries
D004262 DNA Restriction Enzymes Enzymes that are part of the restriction-modification systems. They catalyze the endonucleolytic cleavage of DNA sequences which lack the species-specific methylation pattern in the host cell's DNA. Cleavage yields random or specific double-stranded fragments with terminal 5'-phosphates. The function of restriction enzymes is to destroy any foreign DNA that invades the host cell. Most have been studied in bacterial systems, but a few have been found in eukaryotic organisms. They are also used as tools for the systematic dissection and mapping of chromosomes, in the determination of base sequences of DNAs, and have made it possible to splice and recombine genes from one organism into the genome of another. EC 3.21.1. Restriction Endonucleases,DNA Restriction Enzyme,Restriction Endonuclease,Endonuclease, Restriction,Endonucleases, Restriction,Enzymes, DNA Restriction,Restriction Enzyme, DNA,Restriction Enzymes, DNA
D004279 DNA, Viral Deoxyribonucleic acid that makes up the genetic material of viruses. Viral DNA
D004587 Electrophoresis, Agar Gel Electrophoresis in which agar or agarose gel is used as the diffusion medium. Electrophoresis, Agarose Gel,Agar Gel Electrophoresis,Agarose Gel Electrophoresis,Gel Electrophoresis, Agar,Gel Electrophoresis, Agarose
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000091402 Prohibitins Ubiquitously expressed conserved proteins associated with multiple functions including APOPTOSIS; CELL PROLIFERATION, regulations of various immune and mitochondrial functions, and cellular stress responses. Prohibitin 1 and prohibitin 2 form a ring-shaped complex in the INNER MITOCHONDRIAL MEMBRANE. Prohibitin
D000260 Adenoviruses, Human Species of the genus MASTADENOVIRUS, causing a wide range of diseases in humans. Infections are mostly asymptomatic, but can be associated with diseases of the respiratory, ocular, and gastrointestinal systems. Serotypes (named with Arabic numbers) have been grouped into species designated Human adenovirus A-G. APC Viruses,APC Virus,Adenovirus, Human,Human Adenovirus,Human Adenoviruses
D013045 Species Specificity The restriction of a characteristic behavior, anatomical structure or physical system, such as immune response; metabolic response, or gene or gene variant to the members of one species. It refers to that property which differentiates one species from another but it is also used for phylogenetic levels higher or lower than the species. Species Specificities,Specificities, Species,Specificity, Species
D015183 Restriction Mapping Use of restriction endonucleases to analyze and generate a physical map of genomes, genes, or other segments of DNA. Endonuclease Mapping, Restriction,Enzyme Mapping, Restriction,Site Mapping, Restriction,Analysis, Restriction Enzyme,Enzyme Analysis, Restriction,Restriction Enzyme Analysis,Analyses, Restriction Enzyme,Endonuclease Mappings, Restriction,Enzyme Analyses, Restriction,Enzyme Mappings, Restriction,Mapping, Restriction,Mapping, Restriction Endonuclease,Mapping, Restriction Enzyme,Mapping, Restriction Site,Mappings, Restriction,Mappings, Restriction Endonuclease,Mappings, Restriction Enzyme,Mappings, Restriction Site,Restriction Endonuclease Mapping,Restriction Endonuclease Mappings,Restriction Enzyme Analyses,Restriction Enzyme Mapping,Restriction Enzyme Mappings,Restriction Mappings,Restriction Site Mapping,Restriction Site Mappings,Site Mappings, Restriction
D016678 Genome The genetic complement of an organism, including all of its GENES, as represented in its DNA, or in some cases, its RNA. Genomes
D016679 Genome, Viral The complete genetic complement contained in a DNA or RNA molecule in a virus. Viral Genome,Genomes, Viral,Viral Genomes

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