Among the various restriction sites present on a DNA molecule, the restriction endonucleases prefer specific ones. This site preference may be an inherent property of the restriction endonucleases or may reflect the complexities inherent in the DNA molecule. The site preference of restriction endonucleases can be amplified by the use of intercalators that bind to DNA. This can lead to the production of large and partially cleaved DNA fragments. General protein inhibitors that react with sulfhydryl groups can affect the activities of some restriction endonucleases. This can result in the formation of partially digested DNA fragments. Another approach leading to the formation of large DNA fragments involves base substitution or modification of DNA molecules. New restriction sites can be exposed by relaxing the specificity of some restriction endonucleases. Under conditions of relaxed specificity, the recognition sequence shrinks to the core sequence, which is usually two nucleotides shorter than the normal recognition sequence. When the core restriction sequences are unmasked by relaxation of restriction-endonuclease specificity, the normal restriction sequences inaccessible in some DNAs can be exposed by the prevention of DNA modification. All manipulations described here lead to the formation of DNA fragments that are different (large or new) from normal restriction-endonuclease digestion products. These DNA fragments have potential applications in the mapping of DNA, gene-cloning experiments, and genetic experiments on deletion or substitution.
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