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The publication of the human and other metazoan genome sequences has greatly facilitated positioning and analysis of various genomic functional elements, with initial emphasis on coding sequences. However, a complete functional map of sequenced eukaryotic genomes should include positions of all noncoding regulatory elements. Unfortunately, experimental data on genomic positions of a multitude of regulatory sequences, such as enhancers, silencers, insulators, transcription terminators, and replication origins are very limited—especially at the whole genome level. As most genomic regulatory elements (e.g., enhancers) are generally gene-, tissue-, or cell-specific, the prediction of these elements by computational methods is difficult and often ambiguous. Therefore, the development of high-throughput experimental approaches for identification and mapping of genomic functional elements is highly desirable. Our research is focused on the development of experimental approaches for selection of relatively short functionally active DNA fragments from long genomic sequences. The resulting short fragment libraries can be used further for construction of functional maps, either by direct sequencing-mapping, or with the help of microarrays. Recently, we developed methods for identification of nuclear matrix/scaffold attachment regions, potential insulators, open chromatin regions, and binding sites of nuclear proteins.
humgen.siobc.ras.ru/research/sel.html
The Technique
The technique we describe allows for the identification and mapping of in vitro nuclear protein binding sites within long genomic sequences. Unlike chromatin immunoprecipitation, this technique doesn't require any preliminary knowledge of proteins whose binding sites are to be mapped. The data obtained by the use of this and other techniques (published and unpublished) developed by our group will be integrated into functional maps of long (several hundred kilobases) human genome regions or, in a long run perspective, of whole genomes. The resulting maps of genome regulatory elements will help to understand the mechanisms of large-scale regulation of genome activity.
Identification and mapping of DNA binding proteins target sequences in long genomic regions by two-dimensional EMSA, p. 90.
