A rapid technique for the determination of unknown plasmid library insert DNA sequence directly from intact yeast cells

A polymerase chain reaction (PCR)-based technique is described which allows for the determination of library plasmid insert DNA sequence directly and rapidly from intact yeast cells. Yeast spheroplasts are used to template a PCR reaction to amplify the insert sequence. This PCR product is then purif ied and its sequence directly determined using thermal cycle sequencing. Re adable sequence can reproducibly be obtained from multiple yeast colonies i n just two days. Uses of this technique in yeast two-hybrid screening as we ll as other types of yeast library screens are discussed. Copyright (C) 199 9 JohnWiley & Sons, Ltd.