A major challenge in """"""""bottom-up"""""""" physical mapping is filling in gaps between contigs. We will apply the Branch Capture Reaction (BCR), which was developed in our laboratory, to prepare libraries enriched for the desired clones. Chromosome-walking libraries will be constructed using multiplex methods so that many gaps may be filled at the same time. BCR enables covalent attachment of a DNA partial duplex with a longer, modified, displacer strand and a shorter linker strand to a restriction enzyme cleavage site of a recipient duplex DNA. The reaction is sequence-specific and stringent, with attachment requiring homology between the single-stranded portion of the displacer and the recipient DNA duplex. BCR may be used to label a DNA fragment prior to electrophoresis, mark a fragment for affinity chromatography, or introduce both a selectable marker and a new overhang sequence compatible with a different restriction endonuclease site in a cloning vector. Specifically, BCR and other state-of-the-art methods will be applied toward obtaining a high resolution physical map of human chromosome 9, including both ordered clones and an ordered set of unique sequence tagged sites (STS). The BCR method will be available for collaborative studies of other human chromosomes.
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| Bobovnikova, Y; Kim, S J; Wetmur, J G (1996) Insert selection by BamHI methyltransferase protection in P1 phage-based cloning. Gene 170:39-44 |
| Wetmur, J G; Wong, D M; Ortiz, B et al. (1994) Cloning, sequencing, and expression of RecA proteins from three distantly related thermophilic eubacteria. J Biol Chem 269:25928-35 |
| Wong, D M; Wetmur, J G (1994) Some class-IIS restriction endonucleases can cleave across a three-way DNA junction. Gene 150:63-6 |
