Preparation of high molecular weight plant DNA and its use for artificial chromosome construction

The availability of a substantial amount of high molecular weight DNA is an essential prerequisite for the construction of yeast artificial chromosome (YAC) libraries. Parameters concerning protoplast isolation and DNA extraction have been systematically analysed. Conditions have been established for the obtainement of high molecular weight DNA from Arabidopsis thaliana and Nicotiana plumbaginifolia protoplasts either embedded in agarose plugs or in liquid suspension. Restriction fragments were obtained by partial and total digestion with different endonucleases and separated by PFGE. Ligation of partially EcoRI-digested DNA (range 30-300 kb) followed by transformation of yeast spheroplasts gave rise to YACs with an average size of 60 kb. The introduction of a DNA size-selection step before ligation led to production of YACs in the range of 100-200 kb. Clones up to 460 kb were obtained by blunt-end ligation of pre-selected unrestricted DNA