Sticky ends enzymes

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). 

DNA is ideally suited as a structural material in supramolecular chemistry. It has sticky ends and simple rules of assembly, arbitrary sequences can be obtained, and there is a profusion of enzymes for modification. The molecule is stiff and stable and encodes information. Chapter 10 surveys its varied applications in nanobiotechnology. The emphasis of Chapter 11 is on DNA nanoensembles, condensed by polymer interactions and electrostatic forces for gene transfer. Chapter 12 focuses on proteins as building blocks for nanostructures. 

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. 

Restriction enzymes are sequence-specific in that they cut DNA at specific locations along the nucleotide chain. While some of these enzymes yield "blunt" ends to the resulting DNA fragment, others make staggered cuts in the DNA chain to produce "sticky" ends. Over 250 restriction enzymes are now commercially available. 

Cloning would not be possible without restriction enzymes. DNA chains with a "sticky" end act like molecular "Velcro", thereby enabling two pieces of DNA with complementary nucleotide sequences to be joined together. The linking of the DNA strands is brought about by the enzyme DNAligase which permanently joins the assembled DNA sequences with covalent bonds, thereby producing a recombinant DNA molecule. 

By contrast, other restriction enzymes cut at different portions of a DNA molecule, forming an "offset" break with "sticky" ends. For example, the restriction enzyme known as BamHI cuts between two GG nitrogen bases, but at different parts of the DNA molecule, forming "overhangs or sticky ends. These sticky ends provide locations at which new nitrogen base sequences can be inserted. 

This oligonucleotide contains sequences for one or more restriction sites, and after digestion with the proper restriction enzyme, the whole molecule will now have the proper sticky ends. 

Finally, DNA ligase can be used to join two blunt-ended fragments of DNA. This is not very efficient, however, since the complex of enzyme with two blunt-ended DNA molecules is not stabilized by pairing of sticky ends, and it is therefore unlikely to form. When joining DNA, it is usually more efficient to take advantage of the stability of the complex offered by complementary sticky ends. 

Sticky ends are on strands that are cleaved unevenly that allow the formation of recombinant DNA when they are cleaved by the same restriction enzyme because the ends match. 

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 ... 

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