Restriction endonucleases defined

Terms in bold are defined in the cloning 306 vector 307 recombinant DNA 307 restriction endonucleases 307 DNA ligase 307 plasmid 311... 

One of the major obstacles to molecular analysis of genomic DNA is the immense size of the molecules involved. The discovery of a special group of bacterial enzymes, called restriction endonucleases (restriction enzymes), which cleave double-stranded DNA into smaller, more manageable fragments, has opened the way for DNA analysis. Because each enzyme cleaves DNA at a specific nucleotide sequence, restriction enzymes are used experimentally to obtain precisely defined DNA segments called restriction fragments. 

The most striking specificity in DNA hydrolysis is displayed by the restriction endonucleases which are discussed further in Chapter 26. These fussy catalysts cleave only at points within or close to a defined sequence of several nucleotides in double-stranded DNA. For example, the enzyme EcoR I cuts only at the following palindromic sequence ... 

To handle the mass of existing data, powerful computer programs have been developed and various graphical procedures have also been developed to help the human mind comprehend the results.654 655 One important problem is to define and locate what are called consensus sequences. The problem is best illustrated by examples.654 The cleavage site for the EcoRI restriction endonuclease is GAATTC. There is no ambiguity. In a DNA of random sequence this would be expected to occur by chance in about (1 / 4)6 nucleotides (4 kb). On the other hand, the Hmll restriction endonuclease cleaves within the consensus sequence GTYRAC where Y = C or T and R = A or G. 

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 discovery of restriction endonucleases was of crucial importance in sequencing DNA. These enzymes recognize a specific sequence of four to eight bases in double-stranded DNA and cleave the DNA at a precise point in this sequence. For example, the restriction endonuclease known as AIul cleaves the sequence AGCT between the G and C and the one known as Pstl cleaves the sequence CTGCAG between the A and the G. These restriction endonuclease enzymes provide a reproducible way to produce precisely defined fragments of an appropriate size for sequencing. 

Type II restriction-modification systems differ from their type I and type III counterparts in that the endonuclease and DNA methylase activities are conducted by two separate enzymes (not a single multisubunit complex). The restriction endonuclease cleaves both strands of the DNA duplex within a defined recognition sequence, while the companion DNA methylase methylates a specific base within the same recognition sequence. In contrast... 

An interesting subgroup is represented by the type IIS restriction endonucleases. These enzymes recognize an asymmetric site and cleave this sequence at a defined distance after dimerizing on the substrate (for example FokI). 

The power aind utility of recombinant DNA methods, which include techniques of nucleic acid hybridization, are based on the ability to manipulate DNA in defined ways. These molecular tools are dependent on the commercial availability of a variety of well-characterized enzymes for DNA and RNA modification restriction endonucleases which recognize and cleave specific DNA sequences to create DNA fragments of unique size, DNA and RNA polymerases. 

These enzymes are extraordinarily abundant over 1200 restriction endonucleases had been isolated and characterized by early 1990. Of three classes defined, type II restriction enzymes, which generally cut within their recognition sequences, have found uses in a host of biomedical research and diagnostic applications to be discussed below. Type 1 enzymes cut nonspecifically many nucleotides distal to specific recognition sequences and contain both restriction enzyme and DNA modification (see below) activities on different subunits of multienzyme complexes. Type III restriction enzymes share the multienzyme aspeas of type I enzymes but vary in other properties such as ATPase activity and cofactor requirements. 

The first problem in DNA sequencing is to cleave the enormous DNA chain at predictable points to produce smaller, more manageable pieces, a task accomplished by the use of enzymes called restriction endonucleases. Each different restriction enzyme, of which more than 200 are available, cleaves a DNA molecule at well-defined points in the chain wherever a specific base sequence occurs. For example, the restriction enzyme Alul cleaves between G and C in the four-base sequence AG-CT (Figure 28.12). Note that the sequence is a palindrome, meaning that it reads the same from left to... 

We now return to the question of specificity, the defining feature of restriction enzymes. The recognition sequences for most restriction endonucleases are inverted repeats. This arrangement gives the three-dimensional structure of the recognition site a twofold rotational symmetry (Figure 9.37). 

A PCR restriction enzyme analysis takes advantage of the occurrence of approximately 40 thalassemia mutations that either introduce or remove a restriction endonuclease site. The PCR amplified target sequence is digested using restriction enzymes (enzymes that cleave DNA at particular nucleotide sequences) and the pattern of fragmentation on an agarose gel defines the presence or absence of a particular mutation. 

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