The absence of adenine methylation increases the ligation efficiency of partially filled staggered DNA ends.

Many limitations of plasmid construction due to the restricted compatibility of various staggered DNA ends can be circumvented if the ends are partially filled with dNTP's and Klenow fragment (1). For example, BamHl or Sau3A ends filled with dATP and dGTP retain a GA 5' extension which will ligate with high efficiency to various other partially filled restricted ends, including the 5' TC extension produced by filling Sail or Xhol ends with dTTP and dCTP. A comprehensive list of the different restricted DNA ends that can be joined by using this method has been published (2). The usefulness of the partial fill-in technique has been applied to the construction of genomic libraries because the ends of the vector are prevented from rejoining when the overhangs are partially filled. As a result, libraries can be made that have very little contamination by recircularized vector (3). The lack of vector recircularization has greatly contributed to the usefulness of this technique as applied to routine plasmid construction. However, we have found that DNA ends that are (iam-methylated and partially filled ligate with greatly reduced frequency. Even though the unfilled dam-methylated cohesive ends created by BamHl can be readily ligated to each other, if they are filled to produce a GA extension, they will not efficiently ligate to Sail ends filled to produce a TC extension (see below). The experiment described below indicates that DNA isolated from a dam~ strain of E. coli does not exhibit this problem.