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BREAKTHROUGH TECHNOLOGIES

DNA Sequence-Based "Bar Codes" for Tracking the Origins of Expressed Sequence Tags from a Maize cDNA Library Constructed Using Multiple mRNA Sources¹

Department of Agronomy (F.Q., T.-J.W., P.S.S.) and Mathematics (D.A.A.), Interdepartmental Graduate Program in Bioinformatics and Computational Biology (L.G.), Interdepartmental Genetics Graduate Programs (F.L.), Center for Plant Genomics (P.S.S.), Iowa State University, Ames, Iowa 50011

To enhance gene discovery, expressed sequence tag (EST) projects often make use of cDNA libraries produced using diverse mixtures of mRNAs. As such, expression data are lost because the origins of the resulting ESTs cannot be determined. Alternatively, multiple libraries can be prepared, each from a more restricted source of mRNAs. Although this approach allows the origins of ESTs to be determined, it requires the production of multiple libraries. A hybrid approach is reported here. A cDNA library was prepared using 21 different pools of maize (Zea mays) mRNAs. DNA sequence "bar codes" were added during first-strand cDNA synthesis to uniquely identify the mRNA source pool from which individual cDNAs were derived. Using a decoding algorithm that included error correction, it was possible to identify the source mRNA pool of more than 97% of the ESTs. The frequency at which a bar code is represented in an EST contig should be proportional to the abundance of the corresponding mRNA in the source pool. Consistent with this, all ESTs derived from several genes (zein and adh1) that are known to be exclusively expressed in kernels or preferentially expressed under anaerobic conditions, respectively, were exclusively tagged with bar codes associated with mRNA pools prepared from kernel and anaerobically treated seedlings, respectively. Hence, by allowing for the retention of expression data, the bar coding of cDNA libraries can enhance the value of EST projects.

¹ This research was supported by the National Science Foundation Plant Genome Program (grant no. DBI-9975868 to P.S.S., D.A.A., and others) and by the Iowa Corn Promotion Board (to P.S.S.). This is journal paper number J-19730 of the Iowa Agriculture and Home Economics Experiment Station (Ames; project no. 3409, supported by Hatch Act and State of Iowa funds).

² These authors contributed substantially to this report. F.Q. constructed the bar-coded cDNA library. L.G. conducted the bioinformatic analysis of the EST data and extracted expression data from the bar-coded ESTs.

³ Present address: Department of Statistics, Iowa State University, Ames, IA 50011.

⁴ Present address: Department of Biological Chemistry, University of California at Irvine, Irvine, CA 92697.

^* Corresponding author; e-mail schnable{at}iastate.edu; fax 515-294-5256.

Received April 16, 2003; returned for revision May 19, 2003; accepted June 29, 2003.

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