inosine-triphosphate and 2--deoxy-7-deazaguanosine-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

The enzymatic synthesis of 7-deazapurine nucleoside containing DNA (501 bp) is performed by PCR-amplification (Taq polymerase) using a pUC18 plasmid DNA as template and the triphosphates of 7-deaza-2'-deoxyguanosine (c7Gd), -adenosine (c7Ad) and -inosine (c7Id). c7GdTP can fully replace dGTP resulting in a completely modified DNA-fragment of defined size and sequence. The other two 7-deazapurine triphosphates (c7AdTP) and (c7IdTP) require the presence of the parent purine 2'-deoxyribonucleotides. In purine/7-deazapurine nucleotide mixtures Taq polymerase prefers purine over 7-deazapurine nucleotides but accepts c7GdTP much better than c7AdTP or c7IdTP. As incorporation of 7-deazapurine nucleotides represents a modification of the major groove of DNA it can be used to probe DNA/protein interaction. Regioselective phosphodiester hydrolysis of the modified DNA-fragments was studied with 28 endodeoxyribonucleases. c7Gd is able to protect the DNA from the phosphodiester hydrolysis in more than 20 cases, only a few enzymes (Mae III, Rsa I, Hind III, Pvu II or Taq I) do still hydrolyze the modified DNA. c7Ad protects DNA less efficiently, as this DNA could only be modified in part. The absence of N-7 as potential binding position or a geometric distortion of the recognition duplex caused by the 7-deazapurine base can account for protection of hydrolysis.

Topics: Base Sequence; Chromatography, High Pressure Liquid; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA; Endodeoxyribonucleases; Inosine Triphosphate; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Plasmids; Polymerase Chain Reaction; Tubercidin

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