[5] Reverse Cloning Procedure for Generation of Subclones for DNA Sequencing

This chapter describes an easy, efficient, and reliable method for preparing the subclones of long DNA fragments for sequencing. Reverse cloning is simple; it avoids gel purification or fractionation of DNA fragments, use of linkers, and requires only a standard primer conventionally used for DNA sequencing. Repeated sequencing is minimized, thereby shortening the time of sequencing projects. The reverse cloning procedure depends on the rapid speed of the enzyme. However, a consequence of the high speed of polymerization is the lack of proofreading by the DNA polymerase. The sequencing of the both strands of a specific region of DNA, from independently generated clones, provides the necessary check on the fidelity of the overall DNA sequence. The separate generation of complementary subclones should identify any low-probability errors in subclone sequences.