Chimeric YACs were generated at unreduced rates in conditions that suppress coligation.
Chimerism is a major limitation of current YAC libraries. A method based on partially filled-in ends of restriction fragments was designed to avoid coligation as a possible source of chimeras. Model experiments using plasmid DNA as an insert showed that coligation was clearly avoided by this method. Pilot collections of YACs with an average insert size of 650kb were then constructed with and without the partial fill-in treatment. Starting from a mixture of a equal amounts of human and mouse DNA, none of 108 clones was positive by hybridization with both Alu and B2 probes, again suggesting that coligation was effectively blocked. However, 4 out of 10 clones still hybridized to 2 or more locations by FISH on chromosomes in human metaphase spreads, level similar to that in the clones made without the partial fill-in step. These results strongly suggest that chimeric clones generally arise by a mechanism independent of coligation, presumptively based on recombination.
Wada
M
M
First Department of Biochemistry, Kyushu University School of Medicine, Fukuoka, Japan.
Abe
K
K
Okumura
K
K
Taguchi
H
H
Kohno
K
K
Imamoto
F
F
Schlessinger
D
D
Kuwano
M
M
D000818:Animals; D002460:Cell Line; D018244:Chromosomes, Artificial, Yeast; D004274:DNA, Recombinant; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D051379:Mice; D010957:Plasmids; D012685:Sepharose
D004274:DNA, Recombinant; D012685:Sepharose