Endonuclease specific

Attempts are being made to design semisynthetic restriction endonucleases specific for single-stranded DNA or RNA. For example, an oligonucleotide with a sequence complementary to a sequence adjacent the linkage that is to be cut can be covalently linked to a relatively nonspecific nuclease. Such an enzyme derived from micrococal nuclease cuts a single-stranded chain of either DNA or RNA adjacent to the double-stranded region of the ES complex.839... 

Cleavage/fragmentation Restriction endonucleases Specific proteases Add hydrolysis... 

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). 

Artificial endonucleases, ie, molecules able to cleave double-stranded DNA at a specific sequence, have also been developed. These endonucleases can be obtained by attaching a chemically reactive group to a sequence-specific oligonucleotide. When the oligonucleotide is bound to its complementary sequence, the activation of the reactive group results in double-stranded DNA cleavage. 

Exonucleases. Like the endonucleases they are restriction enzymes which act at the 3 or 5 ends of linear DNA by hydrolysing off the nucleotides. Although they are highly specific for hydrolysing nucleotides at the 3 or 5 ends of linear DNA, the number of nucleotides cleaved are time dependent and usually have to be estimated from the time allocated for cleavage. Commercially available exonucleases are used without further purification. 

The first step in DNA sequencing is to cleave the enormous chain at known points to produce smaller, more manageable pieces, a task accomplished by the use of restriction endonucleases. Each different restriction enzyme, of which more than 3500 are known and approximately 200 are commercially available, cleaves a DNA molecule at a point in the chain where a specific base sequence occurs. For example, the restriction enzyme Alul cleaves between G and C in the four-base sequence AG-CT. Note that the sequence is a palindrome, meaning that the sequence (5 )-AGCT-(3 ) is the same as its complement (3 )-TCGA-(5 ) when both are read in the same 5 — 3 direction. The same is true for other restriction endonucleases. 

Restriction endonuclease (Section 28.6) An enzyme that is able to cleave a DNA molecule at points in the chain where a specific base sequence occurs. 

Table 40-2. Selected restriction endonucleases and their sequence specificities ...

Restriction endonuclease A deoxyribonuclease which cuts DNA at specific sequences which exhibit twofold symmetry about a point. Name derives from the fact that their presence in a bacterial cell prevents (restricts) the growth of many infecting bacteriophages. 

E. Physical Methods and Analytical Techniques.—Nucleotide maps of enzymic digests of DNA have been obtained using the same ionophoretic techniques as have been developed for RNA digests. Pancreatic DNase and Neurospora crassa endonuclease produce very similar maps with E. coli DNA but this technique still awaits the discovery of specific DNases. 

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