Genome profiling
Genome profiling is a biotechnology that acquires genome information without sequencing. It can be used for identification and classification of organisms. It was pioneered by Japanese biophysicist Prof. Koichi Nishigaki and his colleagues at Saitama University in 1990 and later. The term 'DNA profiling' was changed to 'genome profiling' to avoid confusion, as the term 'DNA profiling' had begun to be used for a different technology in the field of forensics. In GP, small fragments of genomic DNA are randomly amplified and the random PCR products are subjected to temperature-gradient gel electrophoresis to generate a species-specific mobility pattern. From this, species identification dots are assigned. This approach is clearly superior because it does not require prior knowledge of any gene sequence. It is clear that random PCR can produce commonly conserved genetic fragments, which make it possible to measure the difference between organisms. The GP method has been successfully applied to a wide range of organisms, from viruses and bacteria to animals and plants, for identification and classification. Its unique merit is in the ultra-high performance to obtain the final results, since GP, in principle, requires only a single random PCR plus μTGGE experiment
Procedure and theory
The GP procedure is outlined in the following stepsRandom PCR
A set of genomic DNA and a single primer are subjected to a modified PCR operated at a lower annealing temperature than conventional PCR, allowing less stable template-primer hybrid structures to initiate the elongation reaction. Random PCR requires only a single short primer, whereas conventional PCR requires two types of primers, and these sequences must be predetermined and specific to each template sequence. The primer pfM12 is known to be used universally for any kind of organism.Although random PCR leads to the generation of DNA fragments that are not intentionally designed, the products are theoretically predictable based on knowledge of the template and primer sequences
Micro-TGGE (μTGGE)
Random PCR products are subjected to temperature-gradient gel electrophoresis to separate fragments by size and melting behavior. As the gel temperature increases, each double-stranded DNA fragment denatures at a specific temperature depending on its sequence. This causes a transition in mobility on the gel, resulting in a specific mobility pattern.Spiddo extraction and analysis
Species identification dots are extracted from the genome profile as initial melting points for DNA bands. The positions of these bands are determined by the DNA sequence. In other words, spiddos are theoretically predictable and can be connected to the template sequence. Therefore, spiddos contain a type of information known as SIOWS, which is unique and essential for GP technology based on DNA melting theory.Genome distance
Using spiddo information, the difference between two genomes can be calculated in terms of pattern similarity score. This parameter has been successfully used for species identification and classification, as well as for measuring the degree of mutation.Applications
GP has been applied to a variety of taxa, including viruses, bacteria, fungi, protozoa, insects, fish, animals and plants. An early study by Kouduka et al. reported the congruence between GP-based clustering and classical, phenotype-based taxonomy for insects, fish, and plants. Further investigation has revealed that insects can be more easily classified using GP than with traditional sequencing-based approaches, such as 18S rDNA sequencing. A GP-based genome database has been proposed and is ongoing, in which organisms are properly located in genome sequence space.GP has also been used to confirm the authenticity of fungal culture collections and to detect irreplaceable samples, such as single-celled protists, Radiolaria and Foraminifera, as well as forensic materials such as body fluids. For scientific purposes, GP has been used to discover continuous mutation of body cells, discriminate leaf origins from ambient trees and determine the family relationships of mice.
Utilizing the concept of genome distance, GP has been successfully implemented to detect mutagenic chemicals. This technology is termed GPMA, in which a test organism, such as the bacterium Escherichia coli, is exposed to mutagenic reagents and investigated for sequence changes using genome distance.