A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7-2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (>20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (Ն98% identity), and 16 clones generated nonexact matches (57%-97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching.
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 of the shotgun sequencing of large fragments, and that the method compares favorably to alternative strategies, such as primer walking.
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