This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bjourson, A J Articles by Cooper, J E Search for Related Content PubMed PubMed Citation Articles by Bjourson, A J Articles by Cooper, J E Agricola Articles by Bjourson, A J Articles by Cooper, J E

 Previous Article  |  Next Article 

Appl Environ Microbiol. 1992 July; 58(7): 2296-2301

Combined subtraction hybridization and polymerase chain reaction amplification procedure for isolation of strain-specific Rhizobium DNA sequences.

Food and Agricultural Microbiology Research Division, Department of Agriculture for Northern Ireland, Belfast.

ABSTRACT

A novel subtraction hybridization procedure, incorporating a combination of four separation strategies, was developed to isolate unique DNA sequences from a strain of Rhizobium leguminosarum bv. trifolii. Sau3A-digested DNA from this strain, i.e., the probe strain, was ligated to a linker and hybridized in solution with an excess of pooled subtracter DNA from seven other strains of the same biovar which had been restricted, ligated to a different, biotinylated, subtracter-specific linker, and amplified by polymerase chain reaction to incorporate dUTP. Subtracter DNA and subtracter-probe hybrids were removed by phenol-chloroform extraction of a streptavidin-biotin-DNA complex. NENSORB chromatography of the sequences remaining in the aqueous layer captured biotinylated subtracter DNA which may have escaped removal by phenol-chloroform treatment. Any traces of contaminating subtracter DNA were removed by digestion with uracil DNA glycosylase. Finally, remaining sequences were amplified by polymerase chain reaction with a probe strain-specific primer, labelled with 32P, and tested for specificity in dot blot hybridizations against total genomic target DNA from each strain in the subtracter pool. Two rounds of subtraction-amplification were sufficient to remove cross-hybridizing sequences and to give a probe which hybridized only with homologous target DNA. The method is applicable to the isolation of DNA and RNA sequences from both procaryotic and eucaryotic cells.

This article has been cited by other articles:

• WINSTANLEY, C. (2002). Spot the difference: applications of subtractive hybridisation to the study of bacterial pathogens. J Med Microbiol 51: 459-467 [Abstract] [Full Text]  
• Sawada, K., Kokeguchi, S., Hongyo, H., Sawada, S., Miyamoto, M., Maeda, H., Nishimura, F., Takashiba, S., Murayama, Y. (1999). Identification by Subtractive Hybridization of a Novel Insertion Sequence Specific for Virulent Strains of Porphyromonas gingivalis. Infect. Immun. 67: 5621-5625 [Abstract] [Full Text]  
• Townsend, K. M., Frost, A. J., Lee, C. W., Papadimitriou, J. M., Dawkins, H. J. S. (1998). Development of PCR Assays for Species- and Type-Specific Identification of Pasteurella multocida Isolates. J. Clin. Microbiol. 36: 1096-1100 [Abstract] [Full Text]