In order to sequence large pieces of DNA, the generation of nested deletions is a commonly used approach. The Erase-a-base kit from Promega contains all the reagents necessary to perform nested deletions for the generation of a DNA fragment library. Based on the original procedure by Henikoff, this protocol specifically digests one strand of DNA (ssDNA) with exonuclease III from a 5’ protruding or blunt-end DNA generated by restriction digestion. To generate fragments of different lengths, exonuclease digestions are performed at a set temperature and time interval in order to make a series of deletions. Removal of the ssDNA is performed with S1 nuclease, via ligation and subsequent transformation into JM109 competent cells. Individual clones can then be picked, mini-prepped and sequenced.
In our laboratory we have used this system successfully with a number of cloned, large pieces of DNA. The most complicated aspect of this protocol is figuring out and performing the restriction digestion required for creating exonuclease-resistant susceptible ends, which the provided manual explains very well. Diagrams are included to explain the overhang associated with particular restriction enzymes. If your plasmid lacks the corresponding DNA cut sites, an alternative is use of two 5’ protruding sites, with one site protected with -phosphorothioate dNTPs (methods and reagents also included). Also, plasmids are provided with this system to allow subcloning to a vector with numerous compatible restriction sites for performing nested deletions.
Following the initial subcloning steps, the protocol is relatively easy to perform. A temperature must be chosen for the exonuclease digestion based on your desired rate of deletion. A useful feature of the kit is the inclusion of a pre-digested plasmid DNA control that allows the user to perform a practice run before using your own DNA. After performing the time points, a useful step which is included in the manual is to run part of the deletion time points on an agarose gel, prior to proceeding with ligation and transformation.
In order to optimize yield when digesting the plasmid, it is advantageous to use the protective cutting enzyme first, followed by the susceptible cutting enzyme. In addition, we obtained consistently better results with column-purified plasmid DNA rather than spin columns. My main recommendation with this system is to carefully plan your experiments before jumping in with your DNA. This includes which restriction enzymes you use to generate the overhangs (cutting sequentionally also helps) and the best temperature for your size of time points. Based on our success, I would recommend this kit for performing nested deletions on various DNA fragments using a variety of plasmids.
Barry Hudson, Ph.D.
Post-Doctoral Fellow
Dept of Surgery
Columbia University