The aim of this study is to develop and streamline efficient approaches for the rapid and accurate sequencing of full-length cDNA inserts, which is becoming increasingly important with the large number of novel genes discovered within the Human Genome Project. A strategy for simultaneous shotgun sequencing of multiple cDNA clones selected from high quality """"""""Soares"""""""" infant brain cDNA library (INIB) will be used to generate the complete sequences of 100-300 unique human cDNA clones. Each clone will be prepared microbially and the inserts will be isolated by digestion with restriction enzymes followed by gel-purification. The shotgun library will be constructed using an improved """"""""double adaptor"""""""" method. cDNA inserts will be combined in pools of 20-100 and concatenated by ligation ot form approximately 30-150 kb DNA fragments. The concatamers will be sheared and randomly cloned to M13 vector. After random sequencing, individual cDNA sequence will be identified at the assembly stage using restriction enzyme sites as """"""""tags"""""""" for the ends of each clone, while remaining gaps and complete double-stranded coverage will be completed by using primer- directed sequencing. This study will provide useful human cDNA sequence information and it will result in streamlining of the methodology to rapidly generate full-length cDNA clone sequences.
| Yu, W; Andersson, B; Worley, K C et al. (1997) Large-scale concatenation cDNA sequencing. Genome Res 7:353-8 |
