DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

9. Full-Length cDNA Sequencing Using Differential Extension with Nucleotide Subsets (DENS)

O. Chertkov¹, C. Naranjo¹, D. Zevin-Sonkin³, H. Hovhanissyan³, A. Ghochikyan³, L. Lvovsky³, A. Liberzon³, M.C. Raja^2,3, and L.E. Ulanovsky^1,2

¹Los Alamos National Laboratory, Los Alamos, NM 87545; ²Argonne National Laboratory, Argonne, IL 60439; and ³Weizmann Institute of Science, Rehovot 76100, Isreal

levy@anl.gov

Upon moving to LANL, we are setting up a full-length cDNA sequencing facility using our technology termed Differential Extension with Nucleotide Subsets (DENS) which 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 pre-synthesized 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.

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. Earlier, in a pilot experiment we used DENS 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 10-4. The plotted distribution showed that base-calls with PHRED values less than 20 occurred only 1% of the time. The 8-mer primers for DENS sequencing were selected using our dedicated software.