Microisolation of the chicken Z chromosome and construction off microclone libraries

Abstract

A simple method was used to adapt a standard light microscope for the collection of chicken Z chromosomes from mitotic-metaphase spreads. The DNA of the collected chromosomes was enzymatically amplified using a partially degenerate primer. The resulting sequences, within a size range of 200–800 bp, were cloned to produce a Z chromosome DNA library, using blunt-end ligation into a SmaI-digested pUC18 plasmid (the SureClone system; Pharmacia, U.S.A.). The microcloning experiments produced 1250 clones; the size range of the cloned inserts was 250–800 bp, with an average of 480 bp (176 clones examined). Using male chicken genomic DNA as a probe, 10 out of 17 randomly selected clones showed strong positive signals on Southern blots, confirming the origin of the inserts as chicken DNA. In addition, the Z-chromosome origin of a selected microclone was verified in a semiquantitative Southern blot hybridization that showed positive signals with intensities that were approximately twice as strong for male (ZZ) as for female (ZW) chicken genomic DNA when the clone was used as a probe. The value of these libraries in further analysis of the chicken Z chromosome is discussed.Key words: microdissection, microcloning, chicken Z chromosome.