Nath K, Azzolina B A
Merck Institute for Therapeutic Research, Rahway, New Jersey 07065.
Gene Amplif Anal. 1981;1:113-30.
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.
在DNA分子上存在的各种限制酶切位点中，限制性内切酶偏爱特定的位点。这种位点偏爱可能是限制性内切酶的固有特性，也可能反映了DNA分子固有的复杂性。限制性内切酶的位点偏爱可以通过使用与DNA结合的嵌入剂来增强。这可能导致产生大片段和部分切割的DNA片段。与巯基反应的一般蛋白质抑制剂会影响某些限制性内切酶的活性。这可能导致形成部分消化的DNA片段。另一种导致形成大片段DNA的方法涉及DNA分子的碱基替换或修饰。通过放宽某些限制性内切酶的特异性，可以暴露新的限制酶切位点。在放宽特异性的条件下，识别序列会缩小为核心序列，该核心序列通常比正常识别序列短两个核苷酸。当通过放宽限制性内切酶的特异性使核心限制序列暴露时，通过防止DNA修饰可以暴露某些DNA中无法接近的正常限制序列。这里描述的所有操作都会导致形成与正常限制性内切酶消化产物不同（大片段或新片段）的DNA片段。这些DNA片段在DNA图谱绘制、基因克隆实验以及缺失或替换的遗传实验中具有潜在应用。
