Method enabling fast partial sequencing of cDNA clones

Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method th at takes advantage of cooperativity between four enzymes to monitor DNA syn thesis. To investigate the feasibility of the recently developed technique for tag sequencing, 64 colonies of a selected cDNA library from human were sequenced by both pyrosequencing and Sanger DNA sequencing. To determine th e needed length for finding a unique DNA sequence, 100 sequence tags from h uman were retrieved from the database and different lengths from each seque nce were randomly analyzed. An homology search based on 20 and 30 nucleotid es produced 97 and 98% unique hits, respectively. An homology search based on 100 nucleotides could identify all searched genes. Pyrosequencing was em ployed to produce sequence data for 30 nucleotides. A similar search using BLAST revealed 16 different genes. Forty-six percent of the sequences share d homology with one gene at different positions. Two of the 64 clones had u nique sequences. The search results from pyrosequencing were in 100% agreem ent with conventional DNA sequencing methods. The possibility of using a fu lly automated pyrosequencer machine for future high-throughput tag sequenci ng is discussed. (C) 2001 Academic Press.