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Publisher Summary This chapter focuses on the cloning of polymerase chain reaction (PCR)-generated DNA containing terminal restriction endonuclease recognition sites. Various sites incorporated into the termini of PCR products have proved difficult to cut with their respective restriction endonucleases. The chapter also describes a variation of this strategy in which primers are used that contain restriction enzyme half-sites. Such sites, if they are palindromic, can be reconstituted by the concatamerization reaction.Information about efficient cloning from terminal restriction enzyme sites is provided. Because the generality and rules governing this terminal transferase-like activity of Taq I polymerase have not been well established, it would be prudent to treat PCR products with T4 DNA polymerase prior to concatamerization by T4 ligase. The procedure described does not require additional nucleotides beyond the restriction endonuclease recognition site. For palindromic restriction endonuclease recognition sites, only half the recognition site need be incorporated at the end of each primer because concatamerization would reconstitute the site. Therefore, the oligodeoxynucleotide primers can contain fewer extraneous nucleotides that do not hybridize to the target DNA sequence. |
