Whole Genome Microarray Hybridization Used To Validate A Pcrbased Genomic Subtraction Protocol

Dept of Agronomy, University of Wisconsin-Madison, 1575 Linden Drive, Madison, WI, USA

In recent work, 3000 genes in a maize endophyte, Klebsiella pneumoniae 342 (Chelius et al. 2000) were discovered by hybridization with a microarray containing nearly all ORFs from E. coli Kl2 (Dong et al. 2001). Those genes unique to strain 342 compared to K12 are now being identified. A PCR-based subtraction hybridization protocol from Clontech (Cat. no. K1809-1 based on work by Akopyants et al. 1998) was used to prepare putative 342-specific sequences following subtraction with E. coli K12 DNA. The putative 342-specific DNA and K12 DNA were labeled with Cy5 and Cy3, respectively. The labeled DNA samples were hybridized on E. coli K12 ORF microarrays (Richmond et al. 1999). Of the 3000 K. pneumoniae 342 genes identified in Dong et al. (2001), 521 (17.36%) of the genes in common between K12 and 342 were still present after subtraction. Among the genes of high (>75%) and intermediate identity (55-75%) between the two strains, 256 (13.52%) and 265 (23.96%) of those genes remained in the sample after subtraction. Sequence analysis of randomly selected 24 clones containing subtracted DNA showed that about 85 to 87.5% of the clones had no or very low homology (less than 50% in DNA level) to E. coli Kl2 DNA. Thus by these two measures, the subtraction protocol provided a 5-6-fold enrichment of the sequences of interest. These experiments illustrate the utility of microarray analysis for the rapid validation of genome subtraction protocols. Sequence analysis of randomly selected 67 clones containing putative 342-specific DNA after subtraction with DNA from a clinical isolate, K. pneumoniae MGH78578 (http://genome.wustl.edu/Projects/bacteria/klebsiella.shtml), showed that about 73% to 77% of these clones (also roughly a 5-fold enrichment) had no or low homology to the clinical strain. A subtraction library by subtracting K. pneumoniae MGH78578 DNA from K. pneumoniae 342 DNA was also constructed. K. pneumoniae 342 specific genes with interest that related to the interaction with maize roots will be discussed.

References:

Akopyants et al. (1998) Proc. Natl. Acad. Sci. USA 95, 13108-13113 Chelius et al. (2000) Appl. Environ. Microbiol. 66, 783-787 Dong etal. (2001) Appl. Environ. Microbiol. 67, 1911-1921 Richmond et al. (1999) Nucleic Acids Res. 27, 3821-3835

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