To the editor:

In the mid-1970s, two simple methods were developed to determine gene sequence1,2. A common feature of these methods is that they require the subcloning of the DNA fragment of interest, for instance into a plasmid and its amplification in Escherichia coli. Assuming a rate of spontaneous mutation of 10−9, this amplification step leads to the presence of several thousand mutated plasmids in 1 μg of plasmid DNA. However, these point mutations do not affect the accuracy of the determined gene sequence as a base substitution occurring at the same position in less than 15% of the population used as template is not detected by currently practiced DNA sequencing methods. This situation fulfils the aim of DNA sequencing, which is to determine the original sequence of a gene and not to detect the mutations occurring in this gene during bacterial amplification.

Nowadays, DNA sequencing is also used as a mean for quality control of plasmid preparations intended for clinical applications (i.e., gene therapy or DNA vaccination). In this case, the aim is not to determine the nucleotide sequence of the original plasmid used to transform bacteria, but to control the quality of a plasmid population obtained after amplification. However, as mentioned above, one of the important features of DNA sequencing methods is that they do not detect the quantitatively minor base substitutions occurring during plasmid amplification. Thus, this leads to the paradoxical situation in which DNA sequencing methods whose characteristic is not to detect base substitutions occurring during plasmid amplification are used precisely to certify the absence of such mutations in plasmid DNA populations obtained after amplification.

The resulting confusion may have dramatic consequences. Even if the high number of mutated plasmids corresponds to a very small percentage of the plasmid population, it may be possible for some mutations to give rise to noxious protein by a gain of a dominant aberrant function, as reported for instance for the tumor suppressor p53. Thus, the control of plasmid DNA sequences intended for clinical use should involve more sensitive methods either specially designed for this aim or able to eliminate mutated DNA. Without such technological improvements, there is a risk that the use of therapeutic DNA for gene therapy or DNA vaccination could lead to the injection of numerous undetectable mutated plasmids with potentially unknown biological properties.