Figure 8-15 DNA enzymatic digestion and gel electrophoresis to separate DNA fragments resulting from the digestion. An example of a nucleic acid recognition site and enzymatic cut produced by £o»Rl, a commonly used endonuclease, is shown in the inset.
causing a break, or cut, in the nucleic acid strand (Figure 8-15).
The number and size of fragments produced by enzymatic digestion depend on the length of nucleic acid being digested (the longer the strand, the greater the likelihood of more recognition sites and thus more fragments), the nucleotide sequence of the strand being digested, and the particular enzyme used for digestion. For example, enzymatic digestion of a bacterial plasmid whose nucleotide sequence provides several recognition sites for endonuclease A, but only rare sites for endonuclease B, will produce more fragments with endonuclease A. Additionally, the size of the fragments produced will depend on the number of nucleotides between each of endonuclease A's recognition sites present on the nucleic add being digested.
The DNA used for digestion is obtained by various methods. A target sequence may be obtained by amplification via PCR, in which case the length of the DNA to be digested is relatively short (e.g., 50 to 1000 bases). Alternatively, specific procedures may be used to
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