Specificity, restriction endonucleases

The sequence of DNA recognized by a specific restriction endonuclease is often palindromic. A palindrome is something that reads the same way backward and forward (Fig. 6-2). The sequence of the bottom strand read in the 5 to 3 direction is the same as that of the top strand read in the 5 to 3 direction. The usual analogy for a verbal palindrome is a sentence that reads the same way backward and forward. Madam, I m Adam is the usual example. It s not exactly the same way for DNA palindromes. The top strand does not read the same from the left as from the right the top strand read from left to right is the same as the bottom strand read from right to left. 

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. 

The calibration of the E. coli genetic map in minutes was a temporary expedient. It was followed by physical maps expressed directly as micrometers of DNA length (total length 1.6 mm) or thousands of nucleotide units (kb). A physical map obtained by restriction enzyme mapping is shown in Fig. 26-4B. To obtain this map DNA fragments were prepared using specific restriction endonucleases (Section E, 1). 

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). 

Cutting DNA at precise locations to yield the DNA segment (gene) of interest by use of sequence specific restriction endonuclease (restriction enzyme). 

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. 

Table 2.6. Recognition sequences of some commonly used six-base-specific 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. 

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. 

Repin V, Puchkova L, Rodicheva E, Vydryakova G. Luminous bacteria - the producers of specific restriction endonucleases. Microbiology 1995 64 751-53. Rodicheva E, Medvedeva S, Vydryakova G, Chugaeva Yu, Kuznetsov A. Gene pool maintenance and prospects for using a special-purpose luminous bacteria collection. Appl Biochem Microbiol 1998 34 75-82. 

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. ... 

D) Total human genomic DNA cut by a specific restriction endonuclease will generate three distinctly separable bands. 

They are all palindromes (ignoring punctuation and spacing in the latter two cases), analogous to palindromic sequences of bases in DNA. Just as the five examples are distinguished by being pronounced differently, different palindromes in DNA are distinguished and acted on by different, very specific restriction endonucleases. 

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

---