DIRECT CLONING OF HUMAN 10Q25 NEOCENTROMERE DNA USING TRANSFORMATION-ASSOCIATED RECOMBINATION (TAR) IN YEAST

The transformation-associated recombination (TAR) procedure allows rap id, site-directed cloning of specific human chromosomal regions as yea st artificial chromosomes (YACs). The procedure requires knowledge of only a single, relatively small genomic sequence that resides adjacent to the chromosomal region of interest. We applied this approach to th e cloning of the neocentromere DNA of a marker chromosome that we have previously shown to have originated through the activation of a laten t centromere at human chromosome 10q25. Using a unique 1.4-kb DNA frag ment as a ''hook'' in TAR experiments, we achieved single-step isolati on of the critical neocentromere DNA region as two stable, 110- and 80 -kb circular YACs. For obtaining large quantities of highly purified D NA, these YACs were retrofitted with the yeast-bacteria-mammalian-cell s shuttle vector ERVI, electroporated into Escherichia coli DH10B, and isolated as bacterial artificial chromosomes (BACs). Extensive charac terization of these YACs and BACs by PCR and restriction analyses reve aled that they are identical to the corresponding regions of the norma l chromosome 10 and provided further support for the formation of the neocentromere within the marker chromosome through epigenetic activati on. (C) 1998 Academic Press.