Odagiri T
Department of Virology, Jichi Medical School, Tochigi, Japan.
Virus Genes. 1994 Jul;8(3):271-4. doi: 10.1007/BF01704520.
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.
在使用改良的T7 DNA聚合酶（测序酶）通过双脱氧法对流感病毒PB2聚合酶基因的cDNA进行核苷酸测序时，启动子区域的序列5'-AGCGAAAGCAGG被误读为5'-AGCGAAACGAGG，即第8和第9位的GC双峰被反向读取。当PB2启动子序列上游的BsmI限制性酶切位点序列被T7 RNA聚合酶的启动子序列替换，以及下游区域被非结构（NS）蛋白基因替换时，也发现了这种误读。这些结果表明，测序酶的误读是PB2启动子区域特有的，与上下游序列无关。然而，当标记混合物中的dGTP被另一种核苷酸类似物dITP替代时，误读并未发生。此外，在核苷酸序列为5'-AGCAAAAGCAGG的NS基因启动子区域未发生反转。由于PB2和NS启动子区域之间的核苷酸差异仅在第四个残基处，即PB2为G，NS为A，因此PB2启动子区域中紧跟三联体AAA的G残基被认为是导致测序酶T7 DNA聚合酶误读的信号。这些发现除了警告测序梯压缩外，还提醒在确定DNA序列时可能存在误读。
