BIOTECHNOLOGY TECHNIQUES Volume 7 No.10 (October 1993) pp.703-706 Received 8th August A FAST AND EFFICIEhT METHOD FOR SCREENING OF NESTED DELETIONS USING PCR Ahmed L. Abdel-Mawgood Faculty of Agriculture, Minia University, Minia, Egypt. Mailing address: 3 1 Saada St, Karmous, Alexandria, Egypt. SUMMARY A procedure is presented for using PCR to screen individual colonies, representing clones obtained after controlled unidirectional exonuclease III digestion, for the sizes of the individual inserts. Many clones can be screened at one time since the plasmid templates used for amplification need not be purified but are prepared from individual colonies by a simple procedure that takes less than 30 minutes. INTRODUCTION Screening of nested deletions is usually achieved by standard cracking procedures (Maniatis et al, 1982). The disadvantage of this method is that the exact size of the cloned DNA will not be known since the purified plasmids isolated by these methods are supercoiled.One of the specialized applications of pGEM-7Zf(+) vector (Promega Corp., Madison, WI) is its use for the production of progressive unidirectional deletions in cloned DNA inserts, allowing efficient sequence analysis of large fragments that also are used for general mutagenesis studies. This plasmid contains both the SP6 and T7 promoters flanking the multiple cloning site within the lac a-polypeptide coding region of the enzyme p galacto- sidase gene (Yanish-Perron et al, 1985). To generate unidirectional deletions, two different restriction sites are needed (Henikoff, 1985). Cleavage at one site produces a 3’ overhang 703