Sticky ligation

Attach Artificial Sticky Ends and Ligate Into a Plasmid... 

In addition to the simplicity of directing its interactions by sticky-ended ligation, there are other advantages to DNA as a medium for topological synthesis. The needs of biotechnology have resulted in extremely convenient automated phos-... 

Figure 6. Solid-support-based protocol for the synthesis of a quadrilateral. Beginning with the support containing a closed junction, alternate cycles of restriction and ligation are performed, always at the position indicated as 1 . Selection of the target product (triangle, quadrilateral, pentalateral,...) is determined by the point at which one chooses to restrict at site 2, exposing a sticky end complementary to that exposed by restriction at site 1. This action corresponds to a strand switch (eliminating a zero node), of the sort shown in Figure 8, below. This is emphasized by the lines of different thickness with which the square catenane is drawn.

In the scission of double-stranded DNA by Ce(iv)/EDTA in the presence of pcPNA 1 /pcPNA 2, several fragments should be formed. In the enzymatic ligation, however, the scission fragment (fragment 1 here), whose sticky end completely fits... 

This step depolymerizes the chain of linkers yielding restriction fragments containing single EcoRl sticky ends at both termini. Before cloning these fragments it is important to remove, as completely as possible, the depolymerized linkers since these would also efficiently ligate into the vector and yield colourless recombinant plaques. Since the vast majority of the restriction... 

Alternatively, if the phosphatase treatment is to be used, the Eco Rl-cut vector can be used for blunt-ended ligation after repair of the EcoRI sticky ends. In practice, this latter procedure appears... 

If the enzyme gives 3 - or 5 -sticky ends which cannot be ligated directly into a site in M13mp7 (e.g. Hhal, Hinfl), the fragment termini can be converted into blunt ends ... 

Seal in a capillary and incubate at 14°C for 6h. Use mixture to transform competent cells (e.g. JM101) and plate out (see Section 4.3.11. below). Count the number of plaques for each ligase concentration and choose the optimum concentration. NOTE Blunt-end ligation requires more ligase than sticky-end ligation of Accf-cleaved vector (2-base overlap, Fig. 4.5.) which requires more than ligation of EcoRI, Pstl or Bam Hi-cleaved vector (4-base overlap). 

Preparation of Blunt-Ended Amplification Fragments. Kinased primers are used in the PCR, and the ends of PCR fragments are flushed with E. coli DNA polymerase I and dNTPs to increase the efficiency of ligation. The fragments are then purified as described for the sticky ended fragments. 

Linkers. Short oligonucleotides that can be ligated (connected) to larger DNA fragments, then cleaved (cut) to yield overlapping cohesive (sticky) ends, suitable for ligation to other DNAs that contain comparable cohesive ends. 

The overhangs thus produced can still hybridize ( anneal ) with each other, even if they came from different parent DNA molecules, and the enzyme ligase will then covalently link the strands. Sticky ends therefore facilitate the ligation of diverse segments of DNA, and allow the formation of novel DNA constructs. 

Figure 7 Cloning by restriction digest and ligation. Both the PCR-amplified gene of interest and the target vector are digested with the same restriction enzymes (or with enzymes that produce compatible ends). The short section of overhanging sequence (called sticky ends ) are complementary to each other. The digested DNAs are mixed, treated with ligase, and transformed into a suitable Escherichia coli host strain to produce a new recombinant DNA molecule with the gene of interest specifically inserted into a host vector.

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