Assessment of clone identity and sequence fidelity for 1189 IMAGE cDNA clones.
This report documents the error rate in a commercially distributed subset of the IMAGE Consortium mouse cDNA clone collection. After isolation of plasmid DNA from 1189 bacterial stock cultures, only 62. 2% were uncontaminated and contained cDNA inserts that had significant sequence identity to published data for the ordered clones. An agarose gel electrophoresis pre-screening strategy identified 361 stock cultures that appeared to contain two or more plasmid species. Isolation of individual colonies from these stocks demonstrated that 7.1% of the original 1189 stocks contained both a correct and an incorrect plasmid. 5.9% of the original 1189 stocks contained multiple, distinct, incorrect plasmids, indicating the likelihood of multiple contaminating events. While only 739 of the stocks purchased contained the desired cDNA clone, agarose gel pre-screening, colony isolation and similarity searching of dbEST allowed for the identification of an additional 420 clones that would have otherwise been discarded. Considering the high error rate in this subset of the IMAGE cDNA clone set, the use of sequence verified clones for cDNA microarray construction is warranted. When this is not possible, pre-screening non-sequence verified clones with agarose gel electrophoresis provides an inexpensive and efficient method to eliminate contaminated clones from the probe set.
Halgren
R G
RG
Department of Biochemistry and Molecular Biology, National Food Safety and Toxicology Center, Michigan State University, East Lansing, MI 48824-1319, USA.
Fielden
M R
MR
Fong
C J
CJ
Zacharewski
T R
TR
D000818:Animals; D001419:Bacteria / Q000302:isolation & purification; D003001:Cloning, Molecular / Q000379:methods; D019295:Computational Biology / Q000379:methods; D018076:DNA, Complementary / Q000032:analysis*; D004340:Drug Contamination; D004587:Electrophoresis, Agar Gel / Q000379:methods; D015723:Gene Library; D014644:Genetic Variation; D008297:Male; D051379:Mice; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods; D012689:Sequence Homology, Nucleic Acid; D013737:Testis
D018076:DNA, Complementary