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Eukaryotic gene invasion by a bacterial mobile insertion sequence element IS2 during cloning into a plasmid vector
Alireza G Senejani1, Joann B Sweasy
1Department of Therapeutic Radiology and Human Genetics, Yale University School of Medicine, New Haven, CT 06520, USA. Joann.sweasy@yale.edu.
Genome Integrity
|August 4, 2010
Summary
Bacterial DNA replication in Escherichia coli (E. coli) can unexpectedly insert novel DNA sequences, like the IS2 element, into cloned genes. Researchers must verify sequences and annotations to prevent errors in genetic databases.
Area of Science:
- Molecular Biology
- Genomics
- Bioinformatics
Background:
- Escherichia coli (E. coli) is a standard host for DNA cloning and gene amplification.
- Maintaining insert integrity during bacterial replication is crucial for accurate genetic studies.
Purpose of the Study:
- To investigate an observed increase in mouse genomic fragment size after E. coli replication.
- To identify the source of the novel DNA sequence within the mouse genomic insert.
- To highlight potential issues with genetic data submission and annotation.
Main Methods:
- Subcloning of a mouse genomic fragment into a plasmid vector.
- DNA sequencing of the amplified insert.
- BLAST (Basic Local Alignment Search Tool) analysis against GenBank.
- Comprehensive search of GenBank for similar sequences and annotations.
Main Results:
- The mouse genomic insert size increased significantly upon replication in E. coli.
- A novel DNA sequence, identified as the Insertion Sequence 2 (IS2) element from E. coli, was found within the insert.
- BLAST searches revealed the IS2 element is present in numerous eukaryotic sequences in GenBank, often incorrectly annotated.
Conclusions:
- Bacterial replication processes, specifically in E. coli, can lead to unintended insertion of mobile genetic elements like IS2.
- The presence of IS2 in eukaryotic sequences within GenBank highlights potential annotation errors.
- Emphasizes the need for rigorous sequence analysis, including BLAST comparisons and annotation verification, before data submission to public databases.
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