DNA-SEQUENCING USING DIFFERENTIAL EXTENSION WITH NUCLEOTIDE SUBSETS (DENS)

Here we describe template directed enzymatic synthesis of unique prime rs, avoiding the chemical synthesis step in primer walking, We have te rmed this conceptually new technique DENS (differential extension with nucleotide subsets), DENS works by selectively extending a short prim er, making it a long one at the intended site only, The procedure star ts with a limited initial extension of the primer (at 20-30 degrees C) in the presence of only two out of the four possible dNTPs, The prime r is extended by 6-9 bases or longer at the intended priming site, whi ch is deliberately selected, (as is the two-dNTP set), to maximize the extension length, The subsequent termination reaction at 60-65 degree s C then accepts the extended primer at the intended site, but not at alternative sites, where the initial extension (if any) is generally m uch shorter DENS allows the use of primers as long as 8mers (degenerat e in two positions) which prime much more strongly than modular primer s involving 5-7mers and which (unlike the latter) can be used with the rmostable polymerases, thus allowing cycle-sequencing with dye-termina tors compatible with Tag DNA polymerase, as well as making double-stra nded DNA sequencing more robust.