Simultaneous Shotgun Sequencing of Multiple cDN A Clones

Abstract

This study describes an efficient method for the rapid sequencing of DNA fragments in the size range 1–5 kb. Individual fragments, here cDNA inserts, are purified by restriction enzyme digestion and gel purification, pooled and concatenated by ligation. The concatamers are sheared and cloned randomly into M13, followed by random sequencing. The sequences of the individual cDNA inserts are obtained at the assembly stage using restriction enzyme sites as ‘tags’ for the ends of each fragment. In this study the sequencing of two libraries containing 7 and 16 cDNA inserts with an average length of 2.5 kb is described. The results show that the efficiency of the procedure is comparable to that of the shotgun sequencing of large fragments, and that the method compares favorably to alternative strategies, such as primer walking.

Key Words:

• Full-length cDNA sequences
• Concatenation
• Shotgun sequencing