Site-specific restriction endonucleases

The discovery of site-specific restriction endonucleases in 1970 and the development of efficient DNA sequencing methods in 1977 sparked a revolution in biology. On Oct 1,1990, the 15-year project to sequence the complete human genome was officially begun. ... 

A restriction map of plasmid pBR322 indicating the positions of its antibiotic resistance genes and the sites of nucleotide sequences recognized by specific restriction endonucleases. 

Figure 26-7 Genetic map of cloning plasmids pBR322 and pUC18. Abbreviations ori, origin of replication Amp1, ampicillin resistance gene Tet1, tetracycline resistance gene. Other abbreviations are for sites cleaved by specific restriction endonucleases, a few of which are defined in Table 26-2. The nucleotide sequence numbers and directions of transcription are also indicated. Reproduced by permission of Amersham Pharmacia Biotech Inc.

The plasmid should have only one cleavage site for a specific restriction endonuclease. This provides only two ends to which the foreign DNA can be attached. Ideally, the single restriction site should be within a gene, so that insertion of the foreign DNA will inactivate the gene (called insertional marker inactivation). 

SPECIFICITY. Restriction endonucleases are highly specific in hydrolyzing doubled-stranded DNA. Not only do they recognize a specific sequence of four to six base pairs (up to 11 base pairs in case of Bgl I see Table II) but the base pairs must be patind/iomic in the two strands of DNA. The cleavage sites possess twofold rotational symmetry as shown in Scheme I for restriction endonuclease Aat II from AcdtobacXdA OUddtL (Table II A, T, G, C indicate deoxyribonucleotides in this paper). In this example, the T-C phosphodiester bond in each strand two residues distal to the symmetry axis is cleaved by restriction endonuclease Aat II. 

Before sequencing, the DNA chain is cleaved at specific sites by restriction endonucleases. 

The most widely used genetic screening technique, PCR-RFLP detects a mutation at a specific restriction endonuclease cleavage site at the mutation locus [9], The products from other techniques such as ARMS (amplification refractory mutation system), SSCP (single-strand conformational polymorphism), HPA (heteroduplex polymorphism), and CDCE (constant denaturant capillary electrophoresis) and PCR are usually separable using polymer networks. 

The recognition sites for restriction endonucleases arc specific palindromic sequences of DNA"" not more than 8 bp long. A number of these palindromes are listed in Table 6-2. A palindrome is a. sequence of letters that reads the same way forward and backward, for instance "A man. a plan, a canal Panama ." "DNA-land," "Did Hannah sec bees Hannah did." Restriction endonucleases cleave DNA at palindromic sites to yield several types of cuts ... 

FIGURE 7.4 In RARE cleavage, specific restriction endonuclease sites are protected with a complementary oligonucleotide. In the presence of RecA, a triplex DNA complex is formed that prevents methylation at the protected site(s), whereas unprotected sites are methylated by the corresponding methylase. After dissociation of the complex, the protected sites can be cleaved with the specific restriction endonuclease. 

Some palindromic sequences of double-stranded DNA recognized by specific restriction endonucleases U. indicates cleavage site) and/or modification enzymes ( indicates methylated... 

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). 

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