RNA Genomic DNA
Restriction Endonuclease Digestion of Genomic DNA
Many applications require conversion of genomic DNA into conveniently sized fragments by restriction endonuclease digestion. Restriction endonucleases are bacterial enzymes that bind and cleave DNA at specific target sequences. Type II restriction enzymes are the most widely used in molecular biology applications. These enzymes bind DNA at a specific recognition site, consisting of a short palindromic sequence, and cleave within this site, for example, AGCT (for AluI), GAATTC (for EcoRI), and so on. Isoschizomers are different enzymes that share the same specificity and in some cases, the same cleavage pattern.
Isoschizomers may have slightly different properties that can be very useful. For example, the enzymes MboI and Sau3A have the same sequence specificities, but MboI does not cleave methylated DNA, while Sau3A does. Sau3A can therefore be used instead of MboI where necessary.
Selecting suitable enzymes The following factors need to be considered when choosing suitable restriction enzymes:
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Fragment size Blunt-ended/sticky-ended fragments Methylation sensitivity Compatiblity of reaction conditions (when carrying out digests with more than one enzyme)
Fragment size Restriction enzymes with shorter recognition sequences cut more frequently than those with longer recognition sequences. For example, a 4 base pair (bp) cutter will cleave, on average, every 44 (256) bases, while a 6 bp cutter cleaves every 46 (4096) bases.
Use 6 bp cutters for mapping genomic DNA, as these give fragments in a suitable size range for cloning.
Blunt-ended/sticky-ended fragments Some restriction enzymes cut both DNA strands at the same position, creating blunt-ended DNA fragments. However, the majority of enzymes make cuts staggered on each strand, resulting in a few base pairs of singlestranded DNA at each end of the fragment, known as “sticky” ends. Some enzymes create...