Restriction enzymes blunt ends

Restriction enzymes are named after the bacterium from which they are isolated. For example, EcoRI is from Escherichia coli, and BamEII is from Bacillus amyloliquefaciens (Table 40-2). The first three letters in the restriction enzyme name consist of the first letter of the genus (E) and the first two letters of the species (co). These may be followed by a strain designation (R) and a roman numeral (I) to indicate the order of discov-ery (eg, EcoRI, EcoRIE). Each enzyme recognizes and cleaves a specific double-stranded DNA sequence that is 4—7 bp long. These DNA cuts result in blunt ends (eg,... 

Biolabs (Pickering, ON), and Promega (Madison, WI). It is important to note that restriction enzymes producing 3 -overhangs should be avoided if possible (e.g., Psfl, Sfil, Kpnl). The use of such enzymes has been reported to result in the production of additional, nonspecific transcripts (Schenborn and Mierendorf, 1985). If these enzymes must be used, an exonuclease such as DNA Polymerase 1 Large (Klenow) Fragment can be utilized to convert the overhang to a blunt end before the template is transcribed. 

Restriction enzymes are sequence-specific in that they cut DNA at specific locations along the nucleotide chain. While some of these enzymes yield "blunt" ends to the resulting DNA fragment, others make staggered cuts in the DNA chain to produce "sticky" ends. Over 250 restriction enzymes are now commercially available. 

Excessive amounts of RNA polymerase should not be used with the vector containing both T3 and T7 promoters. Otherwise, transcription may not be promoter-specific (strand-specific) Nonspecific initiation of RNA transcripts may also occur at the ends of the DNA template. This is most prevalent with a 3 -protrud-mg terminus Nonspecific initiation may be reduced by increasing the final NaCl concentration in the transcription buffer to 100 mM. When possible, restriction enzymes that leave blunt or 5 -protruding ends should be used... 

Digest 1 pg of the pLXSN retroviral plasmid with 1 pi of the restriction enzyme Hpal in 10 pi of 1 x NEBuffer 4 in a 1.5 ml Eppendorf tube at 37°C overnight. This produces a linear 5.9 kb backbone fragment with blunt ends. Add 1 pi of calf intestinal alkaline phosphatase to the tube which removes 5 and 3 phosphoryl groups. This prevents the plasmid from self ligation. 

Restriction enzymes recognize specific recognition sequences and cut the DNA to leave cohesive ends or blunt ends. The ends of restricted DNA molecules can be joined together by ligation to create new recombinant DNA molecules. 

Some restriction enzymes cleave both strands of DNA so as to leave no unpaired bases on both ends known as blunt ends (subsequent joining requires linkers). 

Fig. 10. Incremental truncation libraries (Ostermeier et al., 1999b). Plasmid DNA is digested with two restriction enzymes one that produces a 3 recessed end (A which is susceptible to Exo III digestion) and the other that produces a 5 recessed end (B which is resistant to Exo III digestion). Digestion with Exonuclease III proceeds under conditions in which the digestion rate is slow enough so that the removal of aliquots at frequent intervals results in a library of deletions of all possible lengths from one end of the fragment. The ends of the DNA can be blunted by treatment with SI nuclease and Klenow so that unimolecular ligation results in the desired incremental truncation library.

Figure 15.1 Two types of cleavage made by restriction enzymes (A) cuts symmetrically placed around the line of symmetry to form overlapping cohesive ends (B) cuts on the line of symmetry to form nonoverlapping blunt ends.

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