Vol. 39, No. 4, July 1996 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages 771-779 DESCRIPTION OF A ONE STEP STAGGERED REANNEAL1NG METHOD FOR DIRECTIONAL CLONING OF PCR-GENERATED DNA USING STICKY-END LIGATION WITHOUT EMPLOYING RESTRICTION ENZYMES Menachem Ailenberg and Mel Silverman MRC Group in Membrane Biology, Department of Medicine, University of Toronto, Toronto, Ontario, Canada, M5S 1A8 Received May ! 2 , 1996 A novel method is described for ligation of PCR products into vectors. It utilizes formation of sticky end sequences compatible with those generated on the plasmid. Four primers are used: two primers are designed to contain the desired sequence of the sticky end plus the sequence of the gene and the other two primers contain the same sequence as the first two primers without the additional bases. The primers are paired to create two PCR products containing the additional bases in a staggered 5' position. Following melting and reannealing, the newly formed products contain the additional sequences as sticky overhangs compatible with the sticky ends of the plasmid. Key words: cDNA cloning, PCR, staggered reannealing, ligation INTRODUCTION The ability to readily ligate foreign cDNA into a vector of choice is a key requirement in contemporary experimental molecular biology. Several cloning procedures for achieving this objective have been described and are in common use. The usual PCR- based approach is to employ primers containing 5' sequences of the enzyme recognition sites, followed by digestion with the appropriate restriction enzymes so as to create sticky ends (1). Although this method is generally reliable, there are occasional difficulties and certain problems. For example, for efficient digestion, it is necessary to generate 5' sequences that are longer than the actual sequence recognition site of the enzyme. This makes it necessary to empirically estimate primer length. Also, catalysis of restriction 771 1039-9712/96/040771-09505.00/0 Copyright © 1996 by Academic Press Australia. All rights of reproduction in any form reserved_