2--deoxy-7-deazaguanosine-triphosphate and 2--deoxyinosine-triphosphate

A common problem in automated DNA sequencing when applying the Sanger chain termination method is ambiguous base calling caused by band compressions. Band compressions are caused by anomalies in the migration behavior of certain DNA fragments in the polyacrylamide gel because of intramolecular base pairing between guanine and cytosine residues. To reduce such undesired secondary structures, several modifications of the sequencing reaction parameters have been performed previously. Here, we have applied mixtures of the nucleotide analogs 7-deaza-dGTP and dITP instead of dGTP in the cycle sequencing reaction and in combination with varying buffer conditions. Band compressions were particularly well resolved, and reading length was optimal when a ratio of 7-deaza-dGTP:dITP of 4:1 was used in the in vitro DNA synthesis with AmpliTaq FS DNA polymerase. We conclude that the incorporation of both nucleotide analogs at these particular ratios leads to heterogeneous DNA chains that result in a reduction or elimination of intramolecular base pairing and thus a higher accuracy in the base assignment.

Topics: Base Pairing; Deoxyguanine Nucleotides; DNA, Single-Stranded; Electrophoresis, Polyacrylamide Gel; Inosine Triphosphate; Nucleic Acid Conformation; Sequence Analysis, DNA; Taq Polymerase; Templates, Genetic

Topics: Deoxyguanine Nucleotides; Genes, p53; Inosine Triphosphate; Polymerase Chain Reaction; Protein Structure, Secondary; Sequence Analysis

Topics: Base Sequence; Deoxyguanine Nucleotides; DNA; DNA-Directed DNA Polymerase; Electrophoresis, Polyacrylamide Gel; Inosine Triphosphate; Molecular Sequence Data; Nucleotide Mapping