Investigators' Abstract) The development of a methodology is proposed that may contribute to the rapid physical mapping of human and other genomes. This methodology will accelerate the construction of contig maps, which depict overlapping clones, and provide raw material for sequencing. The investigators' approach is as follows: the DNA of several equivalents of a genomic library is isolated, and using an end fragment of clone A that represents a starting point for the mapping, the overlapping clone B is isolated rapidly using a method based on the Escherichia coli protein RecA. This process, repeated many times over, provides an array of overlapping clones, i.e., a contig map. In the present form of the method, RecA expedites heteroduplex formation between the probe and the homologous target. The probe-target complex is isolated using an oligo (dT) column, since the vector carrying the probe has a built-in stretch of dAs. The eluate from the column is used to generate a highly enriched library which can then be re-screened on a single filter to eliminate any background. The overall aim is to perfect this method and apply it to the ordering of fragments of the human genome. This involves: 1) determining essential features of the probe, i.e., its optimal length and required degree of homology to the target; 2) optimizing the topological requirements of the RecA reaction; 3) improving the probe-target isolation procedure by constructing a column where the oligo (dT) is replaced by a DNA sequence specific to the vector carrying the probe, thus eliminating background generated by the dTs of the column hybridizing with stretches of dAs in the genome; and 4) isolating and using a thermostable RecA to increase efficiency and automate the method. To test this methodology, it will be used to isolate a genomic clone of the HLA-DRalpha gene, and to construct a physical map of an MHC region on chromosome 6. This approach will not only be helpful to the human genome project per se, but may also expedite current efforts to isolate genes involved in inherited diseases for which genetic markers already exist.
| Sun, Y; Rigas, B (1994) Detection of biotinylated nucleic acids directly on agarose gels. Biotechniques 16:782-4 |
