Reverse misreading of a GC doublet by the modified T7 DNA polymerase, Sequenase.
In nucleotide sequencing of the cDNA of the influenza virus PB2 polymerase gene by the dideoxy method using a modified T7 DNA polymerase, Sequenase, the sequence of the promoter region, 5'-AGCGAAAGCAGG, was shown to be misread as 5'-AGCGAAACGAGG, i.e., a GC doublet at positions 8 and 9 was read in reverse. This misreading was also found both when the sequence of BsmI restriction site upstream from the PB2 promoter sequence was exchanged by that of the promoter of T7 RNA polymerase and when the downstream region was substituted with the nonstructural (NS) protein gene. These results indicated that the misreading by Sequenase was attributed specifically to the PB2 promoter region, independent of the upstream and downstream sequences. The misreading, however, did not occur when dGTP in the labeling mixture was substituted with another nucleotide analog, dITP. Furthermore, the reversion did not occur in the NS gene promoter region, where the nucleotide sequence was 5'-AGCAAAAGCAGG. Since the nucleotide difference between the PB2 and NS promoter regions was only at the fourth residue, i.e., G for PB2 and A for PB2 and A for NS, the G residue followed by a triplet AAA in the PB2 promoter region was suggested to be a signal responsible for the misreading by Sequenase T7 DNA polymerase. The findings warns of possible misreading in determining DNA sequences, in addition to compression of the sequencing ladder.
Department of Virology, Jichi Medical School, Tochigi, Japan.
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Odagiri
D017123:Bacteriophage T7; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D054306:Dideoxynucleotides; D005814:Genes, Viral / Q000235:genetics; D009980:Influenza A virus / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012324:RNA-Dependent RNA Polymerase; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D013942:Thymine Nucleotides; D014764:Viral Proteins / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics
D017931:DNA Primers; D004279:DNA, Viral; D054306:Dideoxynucleotides; C057027:PB2 protein, Influenzavirus A; D013942:Thymine Nucleotides; D014764:Viral Proteins; D015678:Viral Structural Proteins; C034970:bacteriophage T7 induced DNA polymerase; D012324:RNA-Dependent RNA Polymerase; D004259:DNA-Directed DNA Polymerase; C017216:2',3'-dideoxythymidine triphosphate