Restriction enzyme digestion

The choice of restriction enzymes is dictated by a number of factors. Often, the choice will be guided by a knowledge of the restriction enzyme sites in and around the gene to be analyzed. Where no such prior knowledge exists, three further considerations must be made. First, the frequency with which a restriction enzyme cuts DNA is a function of the length of the cut-site recognition sequence; the longer the sequence, the less frequently the enzyme will cut. Secondly, in humans, this frequency is reduced if the recognition sequence contains a CpG dinucleotide which occurs less frequently than expected due to a secondary consequence of DNA methvlation. Finally, some restriction enzymes are inhibited depending on whether or not the recognition sequence is methylated. Such methylation is often tissue-specific in nature. For most applications,

DNAs are cur with restriction enzymes producing fragments in the size range 0.5-15 kb.

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