Exonuclease III digestion

Bethesda Research Laboratories. These primers do not require prior Exonuclease III digestion but should be separated from the vector after release. 

Pt binding impedes exonuclease III digestion the stop site is at G-G, adjacent bases showing intrastrand crosslink. 

Additional study of the termini of AAV DNA by Koczot et al. (1973) has shown that AAV DNA also contains an inverted terminal nucleotide sequence repetition of the type found in adenovirus DNA (Garon et al., 1972 WoLFSON and Dressler, 1972). This conclusion was based upon the fact that up to 70% of separated plus or minus strands of AAV DNA formed single-stranded circles when annealed and these circles were converted to linear molecules by exonuclease III digestion. It was hypothesized that such single-... 

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.

Exonuclease III is a 3 - 5 exonuclease which is specific for double-stranded DNA (Richardson et al., 1964). Digestion of a linear duplex proceeds from the 3 -ends of both strands and the products of a complete digest are two single-stranded molecules, each about half the length of the original duplex with probably only a small amount of complementarity remaining between them at their 3 -ends. Provided the exonuclease is free from contaminant activities, these single... 

A linear duplex DNA at a concentration of 5 p.mol in 10 pA of exonuclease III buffer (70 mM Tris-HCl, pH 8.0, 1.0 mM MgCl2, 10 mM dithiothreitol) is degraded at an approximate rate of 500 base pairs/hour/duplex end at 20°C using an enzyme concentration of 0.5 units//d of exonuclease III (Smith, 1980). One unit of enzyme activity is as described by Richardson et al. (1964). Usually the DNA to be sequenced will be a linear duplex of approximately known length derived from a restriction digest. Digestion may be... 

Fig. 4.10. Digestion of the 96 base-pair Ri-Ri universal primer with exonuclease III. Note that the 5 -ends are left intact thus yielding primers with a fixed defined 5 -end. Since the template used in the annealing reaction is single-stranded (the + strand) only one of the two single-stranded products will subsequently anneal to the...

A useful alternative to the T4 DNA pol method is offered by the sequential 3 5 digestion with exonuclease III (exo III) and repair... 

Since the synthesized fragments terminated with dideoxynucleotides, they do not carry free 3 -OH ends. These terminators can be removed by 3 - 5 exonuclease activity of several DNA polymerases or nucleases, resulting in 3 -OH ends. In our case, terminator removal was combined with the reassembly by employing a mixture of thermostable exonuclease 111 and Tag polymerase. Exonuclease III removes the terminators with its 3 - 5 -exonuclease activity, and Tag polymerase extends the digested fragment with its 5 - 3 -polymer-ase activity. 

This assay used the enzyme exonuclease III (32) which digests... 

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