14. Cosmid Finishing and Full Insert cDNA Sequencing Using Differential Extension with Nucleotide Subsets (DENS) 

D. Zevin-Sonkin^1,2, H. Hovhanissyan¹, A. Ghochikyan¹, L. Lvovsky¹, A. Liberzon¹, M.C. Raja^1,3, E. Ben-Asher², G. Glusman², D. Lancet², and L.E. Ulanovsky^1,3 
1Dept. of Structural Biology and ²Dept. of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, ISRAEL; ³CMB, Argonne National Laboratory, Argonne, IL 60439-4833 
levy@anl.gov 

Differential Extension with Nucleotide Subsets (DENS) is essentially primer walking without primer synthesis (Raja et al., 1997, NAR 25, pp. 800-805). DENS works by converting a short primer (selected from a presynthesized library of 8-mers with 2 degenerate bases each) into a long one on the template at the intended site only. DENS starts with a limited initial extension of the primer (at 20 C) in the presence of only 2 out of the 4 possible dNTPs. The primer is extended by 5 bases or longer at the intended priming site, which is deliberately selected, as is the two-dNTP set, to maximize the extension length. The subsequent termination (sequencing) reaction at 60 C then accepts the primer extended at the intended site, but not at alternative sites where the initial extension (if any) is generally much shorter. 

We use DENS for cosmid finishing and have tested it for full insert cDNA sequencing. The templates for cosmid finishing by DENS (7-8 overlapping fragments, ~ 5 kb each) were PCR amplified from the cosmid. The PCR products were made single-stranded using Lambda Exonuclease (Exo-PCR). If one of the two primers in the PCR is phosphorylated, the Exo digestion leaves the opposite strand single-stranded. The 8-mer primers for DENS sequencing were selected using our dedicated software. The DENS approach resulted in approximately a three-fold reduction in cost and time of finishing compared to the strategy used before: additional shotguns combined with custom synthesized primer walking on the whole cosmid and/or PCR fragments. 

DENS primer walking seems to be tailor-made for full length cDNA sequencing, as the absence of the primer synthesis step facilitates closed-loop automation of primer walking with the benefit of unattended operation. In a pilot experiment we used DENS and "Exo-PCR" for sequencing both strands of four cDNA clones containing inserts of 1.9, 2.3, 3.8 and 4.9 kb. The success rate of the DENS sequencing reactions was 72% yielding 27,864 base-calls. The median PHRED quality value was 40, corresponding to the error probability of approximately one per 10,000. The plotted distribution showed that base-calls with PHRED values less than 20 occurred only 1% of the time.