Restriction enzymes cutting rarely

An important step in the development of techniques allowing such a molecular analysis of large regions in mammalian genomes has been the identification of restriction enzymes cutting rarely in mammalian DNA, and therefore able to cleave the mammalian genome into specific fragments of hundreds to thousands of kilo-base pairs. 

The cuts in the two strands are made at the points indicated by the arrows. This one endonuclease will cut almost any DNA into long pieces averaging about 5000 base pairs each. These pieces can in turn be cleaved by other restriction endonucleases to form smaller fragments. Since there are about 2400 of these enzymes known, with 188 different specificities,536 it is possible to cut any piece of DNA down to a size of 100-500 base pairs, ideal for sequencing.537 539 Each fragment has known sequences at the two ends. Some restriction enzymes cleave outside their specific recognition sequence (see Table 26-2). Some recognize 16-nucleotide palindromes and cut at rare sites. 

Note that plasmids exist as circular dsDNA when they occur in bacteria, that is, when they are replicated and transcribed in viw. When plasmids arc extracted from bacteria they also occur as drcular dsDNA, but they can easily be converted to linear dsDNA in a test tube for the purpose of receiving inserts. Special enzymes called restriction enzymes recognize specific and rarely occurring sequences of base pairs and catalyze the hydrolysis of the dsDNA at a specific point within or near the sequence. In this way, a plasmid consisting of several thousand base pairs can be cut at one site only and converted to a single piece of linear dsDNA,... 

PFGE Whole cells are embedded in agarose gel and genomic DNAs are enzymatically purified and restrict digested by rare cutting enzymes in situ. Large restriction fragments (50-1000 kb) are separated by pulse field gel electrophoresis into a characteristic RFLP. The total procedures take several days. 

The assembled scFv (from Protocol 9) is digested with Sfil (located at the 5 end of the heavy chain) and Notl (located 3 of the fight chain) restriction enzymes to allow cloning into the phagemid vector pHEN-1 cut with the same two en ymes (see Protocol 12). For mouse antibod[y scFv, Sfil and Notl sites are used because there are extremely rare cutters. 

Archaeal chromosomes have been shown to be circular in three cases. For Thermococcus celer [12], restriction mapping of the entire chromosome with three rare-cutting enzymes clearly demonstrated circularity. Sulfolobus solfataricus DNA gives two... 

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