In October 2009, GenBank (the NIH database in Bethesda, Maryland U.S.A.) reported the genetic sequence database exceeded 106 billion nucleotide bases in more than 3,000, 000 named organisms 1 . GenBank, along with the European Molecular Biology Laboratory (EMBL-Bank in Hinxton, U.K.), and the DNA Data Bank of Japan (Mishima, Japan) are the three members of the International Nucleotide Sequence Database Collaboration that exchange information daily to ensure a consistent and complete collection of nucleotide sequence information. This database is expected to keep growing exponentially as sequencing is becoming economically more affordable and demand increases. Therefore, to ensure greater integrity of the data appearing in GenBank, further constructive steps to verify the identity of sequences before submission is becoming increasingly important.

Much of the information in GenBank was obtained by first subcloning a fragment of DNA, followed by its amplification and sequencing. During this process Escherichia coli (E. coli) are commonly used as hosts to multiply the gene of interest faithfully. In this study we report how unusual gene invasions during gene cloning in E. coli have caused incorrect annotation of a number of genes and proteins from numerous diverse species.

We are using a gene targeting approach to generate various knock-in mice. Using PCR, a fragment of the mouse genomic DNA that is 4.5 kb in length and harbors the region of interest was amplified, as shown in Figure 1. After digestion with appropriate restriction enzymes the fragment was inserted into a plasmid. During screening of the transformed host, E. coli Xl1-Blue (Stratagene), one of the resulting recombinant plasmids appeared to carry the insert. However, results from multiple restriction digestions and PCR analysis suggested the presence of an extra fragment of DNA within the insert (Figure 1). After sequencing and comparison of the extra DNA fragment with the entire sequence of the cloned PCR product, the results indicated that the extra piece of DNA element, 1.3 kb in length, was not a result of a duplication of any region of the original PCR amplified region. Furthermore, since the E. coli Xl1-Blue host was recombination deficient, it was not expected to occur as a consequence of potential recombination between the insert and other plasmids or the host genomic DNA.

Astonishingly, using nucleotide-nucleotide BLAST and the extra 1.3 kb DNA as a query sequence against the DNA database, we learned that the presence of the unidentified DNA has been reported in many diverse species, as shown in Figure 2. Among the organisms reported to have the identical DNA fragment are members of eukaryotes and bacteria domains. In bacteria, E. coli received the highest number of hits (61); this was followed by Shigella with more than 10 hits. Among eukaryotes, Oryza sativa received the highest number of hits, 11 times; this was followed by Arabidopsis thaliana, Macaca mulatta, and Homo sapiens with 9, 9, and 5 hits, respectively (see Figure 2).

Results from further sequence analysis indicated that the extra DNA was the E. coli insertion sequence element IS2 that likely incorporated itself into the insert during the cloning process. IS2 is a short 1.3 kb DNA sequence 2 that acts like a simple self mobile genetic element 3 . Although the insertion sequence elements often act as genomic parasites they can sometimes cause chromosome rearrangements and produce mutations leading to elimination or adaptation of their host organism 4 . Multiple copy presence of the IS2 was reported in E. coli more than thirty years ago 5 . During the integration of IS2 into the 4.5 kb fragment we used in our studies, a short piece of DNA with a size of six nucleotides (AGAAAG) was duplicated at the end of insertion. We noticed the presence of a similar duplication (5-8 nucleotides) in nearly all of other entries reported into the GeneBank where the presence of IS2 was recognized (see Table 1). As shown in Table 1, there are 11 copies of IS2 that were detected when the IS2 nucleotide sequence was used to search against the entire genome of the E. coli W311 [GenBank: AP009048]. With an exception of one (AACCC), which was also reported in an earlier study 6 , there is no detectable similarity between duplicated regions found in the 11 copies of IS2 in E. coli.

We have observed a phenomenon in the laboratory whereby bacterial IS2 elements are incorporated into eukaryotic genes during amplification of a recombinant plasmid vector transformed into E. coli cells. The presence of the bacterial IS2 element is reported in many complete/partial genomic DNA or cDNA sequences of numerous diverse eukaryotic species including Homo sapiens, Mus musculus, Macaca mulatta, Oryza sativa, Arabidopsis thaliana, and many others submitted into GenBank. The insertion of the IS2 element occurred most likely during replication in E. coli, similar to our study. For example, if you BLAST the IS2 element from E. coli K-12, one of the top hits is GenBank: AK227066.1 Arabidopsis thaliana mRNA for calcium-dependent protein kinase 19 (CDPK19). If you then take the this Arabidopsis sequence and do a blastn, the top three hits are the Arabidopsis CDPK19 that DO NOT contain the IS2 [GI: 145361922, 30687319, and 836941], whereas the next hits belong to IS2 elements in a variety of organisms including E. coli, plants and animals. On some occasions the IS2 DNA sequence is unintentionally claimed to be a host protein or a part of host protein coding region; i.e. [GenBank: BAE99182] from Arabidopsis thaliana or [GenBank: CAI64485] from Oryza stativarotein 7 . Similarly, when the nucleotide sequence of IS1 was used as a query, the results indicated the presence of this genetic mobile element in many genes found in numerous members of Eukaryota (data not shown). The results of this study suggest one must perform additional careful analysis of the BLAST results from cloned sequences, and further verification of gene annotation before submission into GenBank.

The authors declare that they have no competing interests.

AS carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. JS coordinated, helped to design of the study and improved the manuscript. All authors read and approved the final manuscript.