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Nucleic Acids Research, 1989, Vol. 17, No. 9 3425-3433
© 1989

CHEMISTRY

Construction of yeast artificial chromosome libraries with large inserts using fractionation by pulsed-field gel electrophoresis

Rakesh Anand*,+, Alfredo Villasante§ and Chris Tyler-Smith

Department of Biochemistry, University of Oxford South Parks Road, Oxford OX1 3QU, UK

*To whom correspondence should be addressed

Received February 16, 1989. Accepted March 30, 1989.

A method for constructing yeast artificial chromosome (YAC) libraries with large insert sizes is reported. High molecular weight human DNA was partially digested with EcoRI and cloned in the vector pYAC4. When unfractionated DNA was used, the mean YAC size was 120kb. Fractionation by pulsed-field gel electrophoresis using a ‘waltzer’ apparatus to remove small DNA fragments increased the mean YAC size to ~=220kb or ~=370kb depending on the fractionation conditions. Ligated DNA prepared by this method was stable at 4°C and routinely yielded transformation efficiencies of > 700 colonies/µg. It should be possible to extend the method to produce even larger inserts and to use high molecular weight DNA from any source.

+Present addresses: ICI Diagnostics, Gadbrook Park, Rudheath, Northwich, Cheshire CW9 7RA, UK

§Present addresses: Centro de Biologia Molecular (CSIC-UAM), Cantoblanco, 28049 Madrid, Spain


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