%0 Journal Article
%T Eukaryotic gene invasion by a bacterial mobile insertion sequence element IS2 during cloning into a plasmid vector
%A Alireza G Senejani
%A Joann B Sweasy
%J Genome Integrity
%D 2010
%I BioMed Central
%R 10.1186/2041-9414-1-2
%X 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
%U http://www.genomeintegrity.com/content/1/1/2