A new technique for selective identification and mapping of enhancers within long genomic sequences
We report a new experimental method of direct selection, identification, and mapping of potential enhancer sequences within extended stretches of genomic DNA. The method allows simultaneous cloning of a quantity of sequences instead of tedious screening of the separate ones, thus providing a robust...
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| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2008-05-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/000112732 |
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| Summary: | We report a new experimental method of direct selection, identification, and mapping of potential enhancer sequences within extended stretches of genomic DNA. The method allows simultaneous cloning of a quantity of sequences instead of tedious screening of the separate ones, thus providing a robust and high-throughput approach to the mapping of enhancers. The selection procedure is based on the ability of such sequences to activate a minimal promoter that drives expression of a selective gene. To this end a mixture of short DNA fragments derived from the segment of interest was cloned in a retroviral vector containing the neomycin phosphotransferase II gene under control of a cytomegalovirus (CMV) minimal promoter. The pool of retroviruses obtained was used to infect HeLa cells and then to select neomycin-resistant colonies containing constructs with enhancer-like sequences. The pool of the genomic fragments was rescued by PCR and cloned, forming a library of the potential enhancers. Fifteen enhancer-like fragments were selected from 1-Mb human genome locus, and enhancer activity of 13 of them was verified in a transient transfection reporter gene assay. The sequences selected were found to be predominantly located near 5′ regions of genes or within gene introns. |
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| ISSN: | 0736-6205 1940-9818 |