A NEW PAIR OF M13 VECTORS FOR SELECTING EITHER DNA STRAND OF DOUBLE-DIGEST RESTRICTION FRAGMENTS

The strategy of shotgun cloning with M13 is based on obtaining random fragments used for the rapid accumulation of sequence data. A strategy is sometimes needed for obtaining subcloned sequences preferentially out of a mixture of fragments. Shotgun sequencing experiments showed that not all DNA fragments are obtained with the same frequency and that the redundant information increases during the last third of a sequencing project. Particular fragments are obtained more frequently in one orientation, allowing the use of only 1 of the 2 DNA strands as a template for M13 shotgun sequencing. Two new M13 vectors, M13mp8 and M13mp9, were constructed that permit the cloning of the same restriction fragment in both possible orientations. Consequently, each of the 2 strands becomes a (+) strand in a pair of vectors. The fragments to be cloned are cleaved with 2 restriction enzymes to produce a fragment with 2 different ends. The insertion of such a fragment into the vector can occur only in 1 orientation. Since M13mp8 and M13mp9 have their array of cloning sites in an antiparallel order, either orientation for inserting a double-digest fragment can be selected by the choice of the vector.