Different restriction enzyme-generated sticky DNA ends can be joined in vitro.
We describe a simple method for joining the 5'-protruding, single-stranded DNA ends generated by restriction enzymes. The method allows ends with different sequences to be joined and prevents identical ends from being joined. This is accomplished by partially filling the single strands in a controlled reverse transcriptase reaction. Partial filling can create new single-stranded ends that can be ligated to different, partially filled ends. In almost all useful cases, partial filling simultaneously eliminates the self-complementarity of identical ends and thus prevents them from being joined by DNA ligase. Although all possible combinations of partially filled ends were not tested, the tests performed indicate that the method is fairly general. We demonstrate that ends of the same length can be ligated with useful efficiency if they are: 1) one nucleotide long and complementary; 2) two nucleotides long and complementary or have a mismatch (dA:dC) at one position; or 3) three nucleotides long and, in our test, have a dT:dC mismatch at the middle position.
D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D001483:Base Sequence; D011088:DNA Ligases / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D010957:Plasmids; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D013329:Structure-Activity Relationship; D013604:T-Phages / Q000201:enzymology
D004277:DNA, Single-Stranded; D012194:RNA-Directed DNA Polymerase; D004256:DNA Polymerase I; D004262:DNA Restriction Enzymes; D011088:DNA Ligases