Submitted to: Molecular and Cellular Probes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/5/2007
Publication Date: 2/1/2008
Citation: Lin, H., Doddapaneni, H., Bai, X., Yao, J., Zhao, X., Civerolo, E.L. 2008. Acquisition of Uncharacterized Sequences from Candidatus Liberibacter, an unculturable bacteria, using an improved genomic walking methodAcquisition of Uncharacterized Sequences from Candidatus Liberibacter, an unculturable bacteria, using an improved genomic walking method. 22:30-37. Interpretive Summary: We describe an effective laboratory approach to discover previously unknown DNA sequences from an uncultureable bacterium causing citrus Huanglongbing. Using this technique, we successfully obtained new DNA sequences from the citrus Huanglongbing bacterium. These, together with publicly available DNA sequences cluster into five groups and two singlets representing 25,000 non-redundant nucleotide base pairs. Computer analyses of these sequences predict 12 full length genes and 2 partial genes. The genomic sequences obtained in this study provide new information that will facilitate development of new DNA-based detection tools. The technique described in this study also may be applied to other unculturable organisms for which available DNA sequences are limited or for completion of gaps in DNA sequence positioned between known flanking sequences.
Technical Abstract: We describe an effective PCR-based DNA walking approach to discover previously unknown flanking genomic DNA sequences from Candidatus Liberibacter asiaticus, an uncultureable, phloem-limited bacterium causing citrus Huanglongbing. Using this technique, we successfully obtained 8,813 bp of new DNA sequences from three genomic loci of Ca. Liberibacter asiaticus. These, together with current publicly available Ca. Liberibacter sequences, are clustered into five contigs and two singlets representing 24,477 non-redundant base pairs. BLAST annotation predicts 12 full length genes, 2 partial genes and one pseudogene among these sequences. The genomic sequences obtained in this study provide new information regarding the genome of Ca. Liberibacter that will facilitate development of new genome-based detection tools. The technique described in this study also can be employed to acquire new genomic information for other unculturable or fastidious organisms for which available sequences are limited or for filling in sequence gaps between known flanking genomic DNA sequences.