Plant & Animal Genomes XI Conference

January 11-15, 2003
Town & Country Convention Center
San Diego, CA

Poster: Gene Isolation
            

IDENTIFICATION OF GENE-CONTAINING BAC-CLONES FOR THE ISOLATION OF GENE-ASSOCIATED GENOMIC FRAGMENTS OF BARLEY

Institute for Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Gatersleben, Germany

Gene-associated genomic fragments are useful for plant breeding and biotechnology. They can provide markers for biochemical functions as well as promoters and other regulatory sequences. Especially for large cereal genomes like barley with a high content of repetitive DNA the isolation of gene-associated genomic sequences is a tedious and time consuming process. We established tools to screen more than 300,000 clones of a BAC-library of barley (Yu et al. [2000] TAG 101:1093) for single- and low-copy sequences. Besides high-densitiy colony filters which can be used for hybridisation-based screening, especially with heterologous probes, DNA-pools for a PCR-based strategy have been prepared. 1900 pools of BAC-DNA provide the framework to identify all BAC-clones within the library containing a single-copy sequence with approx. 400 PCRs in four successive rounds of screening. The initial screening step involves 90 PCRs on so-called master pools representing the whole BAC-library and analysis of the products by gel electrophoresis. All PCRs for the following rounds of screening are assembled with a liquid handling robot and worklists for the robot are created automatically from the results of gel electrophoresis to minimize handling errors. Currently a database is under development to store and access all relevant screening results. At present, BAC-clones for more than 100 genes of barley have been identified. 46 of the genes are anchored on the genetic map while others were localized to isolate their promoters.