Restriction enzymes specificity

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. 

Figure 7.8 Sequence-specific recognition sites in the major groove of DNA for three restriction enzymes—Eco RI, Bal I, and Sma I. The DNA sequences that are recognized by these enzymes ate represented by tbe color code defined in Figure 7.7.

Only a rather limited number of base pairs is needed to provide unique and discriminatory recognition sites in the major groove. This is illustrated in Figure 7.8, which gives the color codes for the hexanucleotide recognition sites of three different restriction enzymes—Eco Rl, Bal 1, and Sma 1. It is clear that these patterns are quite different, and each can be uniquely recognized by specific protein-DNA interactions. 

Type II restriction enzymes have received widespread application in the cloning and sequencing of DNA molecules. Their hydrolytic activity is not ATP-depen-dent, and they do not modify DNA by methylation or other means. Most importantly, they cut DNA within or near particular nucleotide sequences that they specifically recognize. These recognition sequences are typically four or six nucleotides in length and have a twofold axis of symmetry. For example, E. coU has a restriction enzyme, coRI, that recognizes the hexanucleotide sequence GAATTC ... 

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. 

Nielsen P.E., Egholm M., Berg R. H., Buchardt O. Sequence specific inhibition of DNA restriction enzyme cleavage by PNA. Nucleic Acids Res. 1993 21 197-200. 

Restriction enzymes are named after the bacterium from which they are isolated. For example, EcoRI is from Escherichia coli, and BamEII is from Bacillus amyloliquefaciens (Table 40-2). The first three letters in the restriction enzyme name consist of the first letter of the genus (E) and the first two letters of the species (co). These may be followed by a strain designation (R) and a roman numeral (I) to indicate the order of discov-ery (eg, EcoRI, EcoRIE). Each enzyme recognizes and cleaves a specific double-stranded DNA sequence that is 4—7 bp long. These DNA cuts result in blunt ends (eg,... 

Restriction enzyme An endodeoxynuclease that causes cleavage of both strands of DNA at highly specific sites dictated by the base sequence. 

Sequencing the amplicon is the most conclusive confirmatory technique. The main consideration is that the DNA must be appropriately purified to achieve unambiguous sequencing data. However, sequencing requires expensive laboratory equipment that may not be available in all labs. Sequencing does not depend upon the specificity of a probe, or restriction enzyme, but gives a direct identification of the amplicon of interest. 

It is clear that both intact cell MALDI-TOF and PFGE have their limitations. PFGE analyses probes the chromosomal DNA of microorganisms for variations in the locations of specific restriction enzyme cleavage sites, while MALDI-TOF mass spectrometry of intact cells primarily examines abundant proteins such as ribosomal proteins35 and those associated with or near bacterial cell walls.58 In order for MALDI-TOF to detect a variation, a mutation must lead to noticeable changes in the expression of cell wall—associated... 

Restriction enzymes are highly specific, attacking only certain sequences (generally four or six base pairs). The host protects its own DNA from the action of restriction enzymes by modifying its... 

Restriction fragment length polymorphism (RFLP) Variation between individuals in DNA fragment sizes cut by specific restriction enzymes. RFLPs are usually caused by mutation at a cutting site. 

Restriction enzymes are sequence-specific endonucleases that cut double-stranded DNA at specific sites. 

Most useful restriction enzymes cut DNA at specific recognition sites, usually four to six nucleotides in length. There can be multiple restriction sites for a single endonuclease within a given piece of DNA, there can be only one (a unique restriction site), or there can be none. It all depends on the sequence of the specific piece of DNA in question. 

Cutting with restriction endonucleases is very useful for moving specific pieces of DNA around from place to place. It s also a useful way to name pieces of DNA. For example, a piece of DNA that is cut from a bigger piece of DNA is often named by size and given a surname that corresponds to the two restriction enzymes that did the cutting—the 0.3-kb EcoRI-BamHI fragment. Restriction enzymes themselves are named for the bacterial strains from which they were initially isolated. 

Restriction enzymes that recognize a specific sequence of five nucleotides should cut the DNA, on average, every 45 base pairs (this is the frequency with which a given sequence of five nucleotides would occur by chance), or every 1024 base pairs. As a result, the average size... 

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