Detection and characterization of chimeric yeast artificial-chromosome clones.
Methods for the construction of yeast artificial-chromosome (YAC) clones have been designed to isolate single, large (100-1000 kb) segments of chromosomal DNA. It is apparent from early experience with this cloning system that the major artifact in YAC clones involves the formation of YACs that contain two or more unrelated pieces of DNA. Such "chimeric" YACs are not easily recognized, particularly in libraries constructed from the total DNA of an organism. In some libraries, they have been found to constitute a major fraction of the clones. Here we discuss some of our experiences with chimeric YACs, with particular emphasis on the approaches that we have employed to detect such aberrant clones. In addition, we describe the detailed characterization of one chimeric YAC isolated from a library prepared from total human DNA. The organization of this clone indicates that it formed by in vivo recombination, presumably in yeast, between two Alu sequences located on unrelated segments of human DNA.
Green
E D
ED
Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110.
Riethman
H C
HC
Dutchik
J E
JE
Olson
M V
MV
D000818:Animals; D010582:Bacteriophage lambda; D015825:Chromosomes, Fungal; D002879:Chromosomes, Human, Pair 10; D002897:Chromosomes, Human, Pair 7; D003001:Cloning, Molecular; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004274:DNA, Recombinant / Q000235:genetics*; D005091:Exons / Q000235:genetics; D005453:Fluorescence; D015723:Gene Library; D006801:Humans; D006822:Hybrid Cells; D007438:Introns / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D016324:Sequence Tagged Sites
C505032:CFTR protein, human; D004274:DNA, Recombinant; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator