Abstract
Splicing by directed ligation (SDL) is a method of in-phase joining of PCR-generated DNA fragments that is based on a pre-designed combination of class IIS restriction endonuclease recognition and cleavage sites. Since these enzymes cleave outside of their recognition sites, the resulting sticky end can have any desired sequence, and the site itself can be removed and does not appear in the final spliced DNA product. SDL is based on the addition of class IIS recognition sites onto primers used to amplify DNA sequences. Cleavage of the PCR products results in elimination of the recognition site-containing flanking sequences and leaves the DNA fragments crowned with protruding ends. With careful design of the sticky ends, several segments can be ligated together in a predetermined order in a single reaction. SDL requires fewer rounds of amplification than overlap extension methods, and is particularly useful for creating a series of recombinants that differ in one segment.
Highlights
The essence of genetic engineering lies in the purposeful shuffling of DNA fragments
At the outset of genetic engineering, in vitro recombination of DNA sequences was based on the chemical synthesis of DNA duplexes with predesigned cohesive ends, which provided the structural basis for subsequent ligation
Its main functions were reduced to obtaining fragments which did not exist in nature, or which could not be excised from DNA because suitable restriction sites were unavailable, or to supplying synthetic restriction sites
Summary
The essence of genetic engineering lies in the purposeful shuffling of DNA fragments. The advent of restriction endonucleases (RE) in the early seventies radically changed the situation by allowing long DNA molecules to be cut into defined fragments (reviewed in 4, 5) This proved to be a powerful complement to molecular cloning and in vivo amplification. Its main functions were reduced to obtaining fragments which did not exist in nature, or which could not be excised from DNA because suitable restriction sites were unavailable, or to supplying synthetic restriction sites This situation changed crucially when PCR was developed in the late eighties (6, 7). The problem of joining together DNA fragments of any origin - including PCR products - in a predetermined fashion remained topical It was largely solved by the elegant method of gene splicing by overlap extension (SOE) (9-11). In the course of a search for other approaches, the DNA splicing by directed ligation (SDL) method was proposed (Figure 1; 1215)
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