DNA sequencing and comparative sequence analysis reveal that the Escherichia coli genomic DNA may replace the target DNA during molecular cloning: evidence for the erroneous assembly of E. coli DNA into database sequences.
DNA sequencing and similarity search of databases provide experimental evidence that portions of the host Escherichia coli genome may get ligated into the cloning vector, resulting in clones containing nontargeted inserts. Several lines of evidence suggest that this non-targeted ligation, as observed by us while subcloning troponin I cDNA, is presumably due to a recombination-mediated mechanism by which host DNA replaces the target DNA in the cloning vector. The E. coli genome mapping to 64-65 min and 92.8-00.1 min, the latter containing insertion sequences, appears to be the hotspot regions involved in this process. We examined the possibility that some sequences reported in the databases may also contain genomic sequences of E. coli. A search of current databases revealed that a rat hepatic glutathione transporter cDNA contains a 2.2-kb-long portion of the E. coli genome that has been wrongly assembled into its 5' untranslated and coding regions. In addition, about 30 sequences in databases, including a Yersinia pestis toxin gene, showed relatively high sequence identity with those portions of the E. coli genome that were present in the nonauthentic clones.
Jha
P K
PK
Department of Anatomy & Cellular Biology, Tufts University Health Science Campus, Boston, MA 02111, USA. pjha@opal.tufts.edu
Sarkar
S
S
D000818:Animals; D016477:Artifacts; D001427:Bacterial Toxins / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial; D016208:Databases, Factual; D004926:Escherichia coli / Q000235:genetics*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D051381:Rats; D017422:Sequence Analysis, DNA; D019210:Troponin I / Q000235:genetics; D015010:Yersinia pestis / Q000235:genetics
D001427:Bacterial Toxins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D026901:Membrane Transport Proteins; D019210:Troponin I; C086860:glutathione transporter