The use of modified primers to eliminate cycle sequencing artifacts

Cycle sequencing is the workhorse of DNA sequencing projects, allowing the production of large amounts of product from relatively little template, Thi s cycling regime, which is aimed at linear growth of the desired products, can also produce artifacts by exponential amplification of minor side-produ cts, These artifacts can interfere with sequence determination, In an attem pt to allow linear but prevent exponential growth of products, and thus eli minate artifacts, we have investigated the use of primers containing modifi ed residues that cannot be replicated by DNA polymerase. Specifically, we h ave used primers containing 2'-O-methyl RNA residues or abasic residues. Ol igomers consisting of six DNA residues and 20 2'-O-methyl RNA residues, wit h the DNA residues located at the 3'-end, primed as efficiently as DNA prim ers but would not support exponential amplification. Oligonucleotides conta ining fewer DNA residues were not used as efficiently as primers, DNA prime rs containing a single abasic site located six residues from the 3'-end als o showed efficient priming ability without yielding exponential amplificati on products. Together these results demonstrate that certain types of modif ied primers can be used to eliminate artifacts in DNA sequencing, The techn ique should be particularly useful in protocols involving large numbers of cycles, such as direct sequencing of BAC and genomic DNA.