Novel system for the sequential, directional cloning of multiple DNA sequences

摘要

A method which combines the use of polymerase chain reaction (PCR) or oligonucleotide linkers and restriction enzymes which cleave degenerate or variable recognition site sequences to allow the cloning of multiple DNA sequences into a vector is disclosed. In this invention, a plurality of unrelated DNA sequences may be directionally cloned within a single vector by adding onto the ends of the sequences, restriction sites with specific sequences which are cleaved by corresponding restriction endonucleases which recognize degenerate or variable recognition sites and which generate cohesive ends upon cleavage. The compatibility (or ability to anneal) of the cohesive ends on different DNA sequences is controlled by the choice of the nucleotide sequence within the recognition sequences of the restriction endonucleases, allowing the DNA sequences to be inserted or joined in any desired orientation. These restriction sites may be selectively incorporated onto either or both end of any DNA sequence of interest using oligonucleotidelinkers or using PCR by adding the restriction sites onto the termini of the 5' and/or 3' primers.