28 - Construction and Use of Chromosome Jumping Libraries

Abstract

Publisher Summary This chapter discusses the construction and use of chromosome jumping libraries. As an alternative to the direct cloning of large fragments in yeast artificial chromosome vectors, the construction of chromosome jumping libraries relies on a series of steps carried out before cloning to reduce the size of large DNA fragments. This is done by introducing large internal deletions, followed by cloning the remaining fragments containing the ends of the original fragment in a λ vector. Jumping libraries fall into two major categories: rare-cutter jumping libraries derived from DNA fragments created by complete or close to complete digestion with an enzyme cutting rarely in the genome, and common-cutter jumping libraries constructed from large DNA fragments generated by a very partial cleavage of genomic DNA with a restriction enzyme cutting often in the DNA used. Rare-cutter jumping libraries find a natural counterpart in linking clone libraries because linking clones inherently connect neighboring rare-cutter jumping clones constructed with the same enzyme, forming the basis of directional chromosome jumping. To ensure the high circularization frequency required for the formation of correct jumping clones, and to reduce the frequency of intermolecular ligation events artifactually connecting ends of different DNA fragments, the circularization step must be carried out at a sufficiently low concentration.