Assessment of clone identity and sequence fidelity for 1189 IMAGE cDNA clones

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• The IMAGE Consortium provides mouse cDNA clones for research.
• Ensuring clone accuracy is crucial for downstream applications like microarray construction.

Purpose of the Study:

• To document the error rate within a specific subset of the IMAGE Consortium mouse cDNA clone collection.
• To evaluate the effectiveness of pre-screening methods for identifying and mitigating errors in cDNA clone collections.

Main Methods:

• Isolation of plasmid DNA from 1189 bacterial stock cultures.
• Agarose gel electrophoresis for pre-screening potential contamination.
• Colony isolation and sequencing for verification.
• Similarity searching against the dbEST database.

Main Results:

• Only 62.2% of stocks were uncontaminated and contained correct cDNA inserts.
• 7.1% of stocks contained both correct and incorrect plasmids; 5.9% contained multiple incorrect plasmids.
• Agarose gel pre-screening, colony isolation, and dbEST searching identified an additional 420 usable clones.

Conclusions:

• A high error rate exists in the analyzed IMAGE cDNA clone subset, necessitating the use of sequence-verified clones for cDNA microarray construction.
• Agarose gel electrophoresis is an effective and economical method for pre-screening non-sequence verified clones to remove contaminated samples.