[Construction of nested set of unidirectional deletion of recombinant plasmid DNA for sequencing]

In order to rapidly sequence a batch of large DNA fragments, we developed a method for the construction of nested set of unidirectional deletions. A nested set of unidirectional deletions of the large DNA fragment of a c-type retrovirus gene was successfully constructed by the adoption of this method. The recombinant plasmid DNA was excised by BamHI and SphI at on end of the target DNA to create 5' and 3' overhanging ends. The DNA was digested with Exo III from 5' overhanging end to generate a set of unidirectional deletion. With the use of this method a set of deleted plasmid DNA and the deleted target DNA excised from the deleted constructs were observed on agrose gel electrophoresis. We sequenced all of 24 deleted DNA fragments (forward and reverse orientation). All of them contained overlapping sequences at their ends. These sequences were readily aligned. The results demonstrate this is an efficient method for sequencing large DNA fragment. The influential factors in this method were discussed in this report.