Restriction enzymes exonucleases

Exonucleases. Like the endonucleases they are restriction enzymes which act at the 3 or 5 ends of linear DNA by hydrolysing off the nucleotides. Although they are highly specific for hydrolysing nucleotides at the 3 or 5 ends of linear DNA, the number of nucleotides cleaved are time dependent and usually have to be estimated from the time allocated for cleavage. Commercially available exonucleases are used without further purification. 

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. 

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.

Klenow polymerase T4-DNA polymerase Exonuclease III Restriction enzymes... 

A specific deletion can be produced by cleaving a plasmid at two sites with a restriction enzyme and ligating to form a smaller circle. This simple approach usually removes a large block of DNA. A smaller deletion can be made by cutting a plasmid at a single site. The ends of the linear DNA are then digested with an exonuclease that removes nucleotides from... 

Another isothermal amplification technique is strand displacement amplification (SDA). " After heat denaturation of DNA in the presence of four primers, dCTP, dGTP dUTP, and a modified deoxynucleotide (dATPaS), two enzymes are added, an exonuclease-deficient polymerase and a restriction enzyme. The two flanking primers that enter into exponential amplification have a restriction site added to their 5 end and get nicked by the restriction enzyme, allow-ing displacement of strands that can in turn be primed, extended, and nicked. Deoxy-ATPotS is used so that the restriction sites include a hemiphosphorothioate linkage to allow single-strand nicking, instead of cutting through double strands. 

The 3 -ends are labeled, after specific restriction enzymes have produced a protruding 5 -end, by a fill-in reaction that allows only one label to be introduced (e.g., labeling with dATP with an overhang with 2 Ts should be avoided). Reverse transcriptase or Sequenase are the preferred enzymes for this fill-in although the Klenow fragment has been widely used. The 3 -> 5 exonuclease activity of Klenow, however, may remove the protruding template end. 

Two clones, carrying a 5.2 kb insert in inverse orientations were isolated and mapped by restriction enzyme analysis. The two clones were used to construct deletion clones with the exonuclease Ill/mung bean nuclease system. Deletion clones containing the psbG homologons region were sequenced. 

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