"half-YAC" Strategy

Multiple strategies were used to identify clones for the most distal unique regions of each human chromosome. First, a "half-YAC" cloning strategy, which was successfully developed in several laboratories, was used (Cross et al., 1989; Riethman et al., 1989). Unlike the conventional YAC cloning procedures in which the vector contains a yeast centromere and two yeast telomeres to stabilize the artificial chromosome, the "half-YAC" strategy enables the cloning of mammalian telomeric DNA by omitting one of the yeast telomeres in the cloning vector. Therefore, the only stable clones that remain are those with human inserts that carry their own natural telomere sequences.

Initially, the majority of clones isolated internal to the half-YAC clones still contained subtelomeric repeats which showed cross-hybridization to other chromosomes by FISH. However, by using the sequence from the vector-insert junction of these half-YACs as the starting point for screening genomic libraries, telomeric clones that were closer to the transitional zone from repetitive to unique DNA were identified. Some of the clones isolated using the vector-insert junction sequences still showed cross-hybridization by FISH. In these cases, "chromosome walking" was used to identify more proximal chromosome specific subtelomere clones.