Bjourson, AJ, Stone, CE and Cooper, JE (1992) COMBINED SUBTRACTION HYBRIDIZATION AND POLYMERASE CHAIN-REACTION AMPLIFICATION PROCEDURE FOR ISOLATION OF STRAIN-SPECIFIC RHIZOBIUM DNA-SEQUENCES. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 58 (7). pp. 2296-2301. [Journal article]
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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 by. 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 P-32, 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.
|Item Type:||Journal article|
|Faculties and Schools:||Faculty of Life and Health Sciences|
Faculty of Life and Health Sciences > School of Biomedical Sciences
|Research Institutes and Groups:||Biomedical Sciences Research Institute|
|Deposited By:||Mrs Caroline Adams|
|Deposited On:||07 Dec 2009 12:12|
|Last Modified:||24 Jun 2011 12:14|
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