Certain endonucleases—enzymes that cut DNA at specific DNA sequences within the molecule—(as opposed to exonucleases, which processively digest from the ends of DNA molecules in a primarily sequence-independent fashion)—are a key tool in recombinant DNA research. These enzymes were termedrestriction enzymes, orREs, because their presence in a given bacterium restricted, or prevented the growth of certain bacterial viruses (bacteriophages). Restriction enzymes cut DNA of any source into unique pieces in a sequence-specific manner—in contrast to most other enzymatic, chemical, or physical methods, which break DNA randomly. These defensive enzymes (hundreds have been discovered) protect the host bacterial DNA from the DNA genome of foreign organisms (primarily infective phages) by specifically inactivating the invading phage DNA by digestion. The viral RNA inducible interferon system provides the same sort of molecular defense against RNA viruses in mammalian cells. However, restriction endonucleases are present only in cells that also have a companion enzyme that site-specifically methylates the DNA of the bacterial host, thereby rendering it noncleavable by that particular restriction enzyme. Thus, sequence-specific DNA methylases and sequence-specific restriction endonucleases that target the exact same sites always exist in pairs in a given bacterium.

Restriction enzymes are named after the bacterium from which they are isolated. For example, EcoRI is from Escherichia coli, and BamHI is from Bacillus amyloliquefaciens (Table 1). 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) in the case of the restriction enzyme EcoRI derived from E. coli strain R. These designations may be followed by a strain designation (R) and a roman numeral (I) to indicate the order of discovery (eg, EcoRI and EcoRII). Each enzyme recognizes and cleaves a specific double-stranded DNA sequence that is typically 4- to 8-bp long. These DNA cuts result in blunt ends (eg, HpaI) or overlapping (sticky or cohesive) ends (eg, BamHI) (Figure 1), depending on the mechanism used by the enzyme. Sticky ends are particularly useful in constructing hybrid or chimeric DNA molecules. If the four nucleotides are distributed randomly in a given DNA molecule, one can calculate how frequently a given enzyme will cut a length of DNA. For each position in the DNA molecule, there are four possibilities (A, C, G, and T); therefore, a restriction enzyme that recognizes a 4-bp sequence cuts DNA, on average, once every 256 bp (44), whereas another enzyme that recognizes a 6-bp sequence cuts once every 4096 bp (46). A given piece of DNA has a characteristic linear array of sites for the various enzymes dictated by the linear sequence of its bases; hence, a restriction map can be constructed. When DNA is digested with a particular enzyme, the cut ends of all the fragments have the same DNA sequence. The fragments produced can be isolated by electrophoresis on agarose or polyacrylamide gels (see the discussion of blot transfer, later); this is an essential step in DNA cloning as well as various DNA analyses, and a major use of these enzymes.

Table1. Selected Restriction Endonucleases & Their Sequence Specificities

Fig1. Results of restriction endonuclease digestion. Digestion with a restriction endonuclease (arrows) can result in the formation of DNA fragments with sticky, or cohesive, ends (A), or blunt ends (B); phosphodiester backbone, black lines; interstrand hydrogen bonds between purine and pyrimidine bases, blue. Generating fragments whose ends have particular structures (ie, blunt, cohesive) is an important consideration in devising cloning strategies.

A number of other enzymes that act on DNA and RNA are an important part of recombinant DNA technology. Many of these are referred to in this and other chapters (Table 2).

Table2. Some of the Enzymes Used in Recombinant DNA Research

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مواضيع ذات صلة

Abnormalities of Chromosome Structure

Impact of Genomic Variation

Variation in individual Genomes

Variation in gene Expression and its relevance to medicine

Allelic Imbalance in gene expression

Gene expression as the integration of Genomic and Epigenomic signals

Epigenetic and Epigenomic aspects of Gene expression

DNA Microarray Technology

RNA Splicing

Initiation of Transcription

Fundamentals of Gene Expression

Cell cycle checkpoints