Figure 3
From: Design and cloning strategies for constructing shRNA expression vectors
Loop digestion can be used to successfully determine the sequence of hairpin templates that are refractory to ordinary sequencing techniques. (a) Strong secondary structure predicted to form in the vector template used for sequencing can 'block' chain elongation, thus terminating the reaction. (b) This can be overcome by first digesting the template within the loop encoding region and sequencing half the template from the forward direction, and (c) the other half from the reverse direction. A small degree of sequence overlap between the forward and reverse reactions, at the restriction site (shown in bold), ensures that every position of the template can be verified.