Ulster University Logo

Ulster Institutional Repository

COMBINED SUBTRACTION HYBRIDIZATION AND POLYMERASE CHAIN-REACTION AMPLIFICATION PROCEDURE FOR ISOLATION OF STRAIN-SPECIFIC RHIZOBIUM DNA-SEQUENCES

Biomedical Sciences Research Institute Computer Science Research Institute Environmental Sciences Research Institute Nanotechnology & Advanced Materials Research Institute

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]

Full text not available from this repository.

URL: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC195771/pdf/aem00048-0202.pdf

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 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
Biomedical Sciences Research Institute > Stratified Medicine
ID Code:1955
Deposited By:Mrs Caroline Adams
Deposited On:07 Dec 2009 12:12
Last Modified:14 Oct 2013 15:53

Repository Staff Only: item control page