Restriction endonucleases sticky ends

As the second educt (B), the plasmid ONA with complementary sticky ends is prepared separately. In the first step the isolated plasmid DNA is cut open by a special type of enzyme called restriction endonuclease. It scans along the thread of DNA and recognizes short nucleotide sequences, e.g., CTGCAG, which ate cleaved at a specific site, e.g., between A and G. Some 50 of such enzymes are known and many are commercially available. The ends are then again extended witfa he aid of a terminal transferase by a short sequence of identical nucleotides complementary to the sticky ends of educt (A). 

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.

This activity is intended to be performed in conjunction with Experiment 66. Restriction endonucleases, or restriction enzymes, cleave DNA at specific base sequences, fragmenting the DNA into smaller pieces. The two strands of a DNA double helix are cleaved at different places, resulting in uneven fragments called sticky ends. Cleavage of DNA by restriction enzymes is a required first step in various types of DNA analysis, including DNA fingerprinting and recombinant DNA technology. 

Cut with restriction endonuclease to generate sticky ends... 

FIGU RE 3.12 Using sticky ends generated by the action of a restriction endonuclease to recombine DNA sequences. 

Some restriction endonucleases make staggered cuts on the two DNA strands, leaving two to four nucleotides of one strand unpaired at each resulting end. These unpaired strands are referred to as sticky ends (Fig. 9-3a), because they can base-pair with each other or with complementary sticky ends of other DNA fragments. Other restriction endonucleases cleave both strands of DNA at the opposing phosphodiester bonds, leaving no unpaired bases on the ends, often called blunt ends (Fig. 9-3b). 

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 ... 

The MCS is a stretch of unique restriction endonuclease cleavage sites between the promoter and the poly (A) signal in expression plasmid vectors, and is used as a common area to insert a cDNA or gene of interest. By using the MCS, the genes of interest can be inserted and assayed with ease. Sticky-end and blunt-end vector strands are available at different restriction sites. Although the MCS is very convenient, it should be noted that these sites usually remain in the mRNA transcribed from the expression unit of the vector and may reduce its stability or the efficiency of translation. Furthermore, one must be careful not to produce an ATG codon at the junctional sequence between the MCS and the insert, because it may work as a false start codon in the resulting mRNA and interfere with the production of the correct product. 

Fig. 13.2. Cleavage of the specific recognition sites by the type II restriction endonucleases EcoRV, EcoRI and Bg/I The cleavage reaction, which requires Mg2+ as cofactor, leads to 5 phosphate and 3 OH ends. While EcoRV cleavage results in blunt ends, EcoRI and Bg/I generate sticky ends with a 5 and 3 overhang, respectively.

Restriction endonucleases are enzymes isolated from bacteria that cut DNA at specific sequences. In this example the enzyme EcoRI (obtained from E. coli) makes staggered cuts that result in the formation of sticky ends. Some restriction enzymes make blunt cuts. ... 

Restriction enzymes (endonucleases) typically cut DNA at specific 4- to 8-bp palindromic sequences, producing defined fragments that often have self-complementary single-stranded tails (sticky ends). 

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