Cohesive ends

Enzyme Common Isoschizomers Recognition Sequence Compatible Cohesive Ends... 

Endonuclease cleavage at the sites indicated by arrows leaves 12-bp cohesive ends, (a-d. Adapted from Figure 1.10.7 in Ansnbel, F. M., et al., eds., 1987. CurreiiL Protocols in Molecular Biology. New York John Wiley Sons e, from Figure 4 in Mnrialdo, H., 1991. Amual Review of Biochemistry 60 136.)... 

FIG. 13 Synthetic DN A motifs for the construction of DNA framework Three- [75] (13) and four-arm (14) DNA junction [8] DNA double-crossover (DX) molecules 15,16 were used for initial studies of enzymatic oligomerization [79]. The DX motif 17, containing four cohesive ends of individual nucleotide sequence, was used for the construction of two-dimensional DNA crystals [80]. 

FIG. 14 Construction of periodic framework and geometric objects from DNA. (a) Construction of two-dimensional DNA lattices from tetravalent four-arm DNA junctions (14) [8]. (b) Synthesis of a macrocyclic molecule from bivalent three-arm DNA junctions (13a) containing two cohesive ends [83]. For simplification, linear double-helical stretches are represented by parallel lines. 

Figure 40-2. Results of restriction endonuclease digestion. Digestion with a restriction endonuclease can result in the formation of DNA fragments with sticky, or cohesive, ends (A) or blunt ends (B).This is an important consideration in devising cloning strategies.

After ethanol precipitation, digest the pTDl vector with another restriction endonuclease to produce the same cohesive end as that introduced in the C-terminal primer. 

Cohesive ends of two DNA fragments form hydrogen bonds. 

The cloning and manipulation of genes usually depends upon the precise cutting of DNA into discrete fragments by restriction endonucleases. Many restriction enzymes generate cohesive ends (sticky ends). Thus, EcoRI produces DNA fragments with the single-stranded "tails" shown here at the 5 -ends of the cut duplexes ... 

These cohesive ends can be used to join together different restriction fragments. It is easy to see that the complementary single-stranded tails can form base pairs to regenerate the original hexanucleotide... 

Two enzymes (Kpnl and Pst in the list in Table 26-2) form 3 -cohesive ends rather than 5 -cohesive ends. In addition, there are three (Alul, EcoRV, and HaellT) that cut at the local twofold axis they form no cohesive ends but leave blunt ends (flush ends). Blunt end fragments are also much used in genetic engineering. "Linkers" that provide cohesive ends can be added.119 The Sfil endonuclease cuts between two 4-bp palindromes in a 13-bp recognition sequence (Table 26-2).120... 

A problem with DNA fragments with cohesive ends is that they spontaneously form closed circles, a process that may compete with a desired joining to another piece of DNA. One solution to this problem is to hydrolyze off the 5 -phosphate groups with an alkaline phosphatase (Chapter 12). This prevents formation of covalently closed circles. However, pieces of DNA that retain their 5 -phosphate groups... 

T4 DNA ligase E. coli carrying an engineered A. phage Ligation of DNA, either blunt or cohesive ends uses ATP... 

E. coli DNA ligase E. coli Ligation of DNA with cohesive ends uses NAD+ as energy source... 

Points m and m are joined in the replicative and prophage forms and bear cohesive ends in the mature phage... 

Insertion of a DNA fragment into a linearized plasmid using cohesive ends and DNA ligase. 

The site of action of EcoRI is a specific hexanucleotide sequence. Two phosphodiester bonds are hydrolyzed (see arrows), resulting in fragmentation of both strands. Note the twofold rotational symmetry feature at the recognition site and the formation of cohesive ends. The weak base pairing between the cohesive ends is not sufficient to hold the two fragments together. 

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

Cuts on the positions symmetrically placed around the line of symmetry to generate cohesive-end (or sticky-end) molecules. 

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