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. This cycling regime, which is aimed at linear growth of the desired products, can also produce artifacts by exponential amplification of minor side-products. These artifacts can interfere with sequence determination. In an attempt to allow linear but prevent exponential growth of products, and thus eliminate artifacts, we have investigated the use of primers containing modified residues that cannot be replicated by DNA polymerase. Specifically, we have used primers containing 2'- O -methyl RNA residues or abasic residues. Oligomers consisting of six DNA residues and 20 2'- O -methyl RNA residues, with the DNA residues located at the 3'-end, primed as efficiently as DNA primers but would not support exponential amplification. Oligonucleotides containing fewer DNA residues were not used as efficiently as primers. DNA primers containing a single abasic site located six residues from the 3'-end also showed efficient priming ability without yielding exponential amplification products. Together these results demonstrate that certain types of modified primers can be used to eliminate artifacts in DNA sequencing. The technique should be particularly useful in protocols involving large numbers of cycles, such as direct sequencing of BAC and genomic DNA.
Stump
M D
MD
Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA. stump@acaris.com
Cherry
J L
JL
Weiss
R B
RB
D016477:Artifacts; D001483:Base Sequence; D017931:DNA Primers; D006358:Hot Temperature; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods
D017931:DNA Primers