Restriction enzymes cohesive ends

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 ... 

Restriction enzymes recognize specific recognition sequences and cut the DNA to leave cohesive ends or blunt ends. The ends of restricted DNA molecules can be joined together by ligation to create new recombinant DNA molecules. 

Figure 15.1 Two types of cleavage made by restriction enzymes (A) cuts symmetrically placed around the line of symmetry to form overlapping cohesive ends (B) cuts on the line of symmetry to form nonoverlapping blunt ends.

In massive parallel signature sequencing (13), 3 sequences of each transcript are isolated using biotinylated primer in the cDNA synthesis and are cleaved with a restriction enzyme that generates a cohesive end. Next, the 3 signature sequences are... 

This cohesive-end method for joining DNA molecules can be made general by using a short, chemically synthesized DNA linker that can be cleaved by restriction enzymes. First, the linker is covalently joined to the ends of a DNA... 

Figure 6.11. Joining of DNA Molecules by the Cohesive-End Method. Two DNA molecules, cleaved with a common restriction enzyme such as EcoBl, can be ligated to form recombinant molecules.

In another valuable approach, cassette mutagenesis, plasmid DNA is cut with a pair of restriction enzymes to remove a short segment (Figure 6.37). A synthetic double-stranded oligonucleotide (the cassette) with cohesive ends that are complementary to the ends of the cut plasmid is then added and ligated. Each plasmid now contains the desired mutation. It is convenient to introduce into the plasmid unique restriction sites spaced about 40 nucleotides apart so that mutations can be readily made anywhere in the sequence. 

The staggered cuts made by this enzyme produce complementary single-stranded ends, which have specific affinity for each other and hence are known as cohesive or sticky ends. Any DNA fragment can be inserted into this plasmid if it has the same cohesive ends. Such a fragment can be prepared from a larger piece of DNA by using the same restriction enzyme as was used to open the plasmid DNA (Figure 5.10). 

Cohesive ends are produced when these terminal extensions are cut by an appropriate restriction enzyme. Thus, cohesive ends corresponding to a particular restriction enzyme can be added to virtually any DNA molecule. We sec here the fruits of combining enzymatic and synthetic chemical approaches in crafting new DNA molecules. 

DNA fragments can be joined (G3) to create artificially recombinant molecules. There are currently three methods for joining DNA fragments in vitro. The first of these capitalizes on the ability of DNA ligase to join covalently the annealed cohesive ends produced by certain restriction enzymes. The second depends upon the ability of DNA ligase from phage T4-infected E. coli to catalyze the formation... 

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