Subtraction Hybridization in Microplates:An Improved Method to Generate Strain-specific PCR Primers

• Published in: Systematic and applied microbiology Vol. 24; no. 1; pp. 108 - 115
• Main Authors: Zwirglmaier, K., Wassill, L., Ludwig, W., Schleifer, K.H.
• Format: Journal Article
• Language: English
• Published: Jena Elsevier GmbH 01.04.2001; Elsevier
• Subjects: Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; diagnostic PCR; DNA Primers; Food Microbiology; Fundamental and applied biological sciences. Psychology; Genetics; Lactococcus lactis; Lactococcus lactis - genetics; Lactococcus lactis - isolation & purification; Listeria monocytogenes; Listeria monocytogenes - genetics; Listeria monocytogenes - isolation & purification; Microbiology; Microplates; Nucleic Acid Hybridization - methods; Pediococcus; Pediococcus - genetics; Pediococcus - isolation & purification; Polymerase Chain Reaction - methods; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - isolation & purification; Species Specificity; subtraction hybridization; subtraction hybridization; Microplates; diagnostic PCR; Evaluation; Lactococcus lactis; Primer; Immobilization; Listeria monocytogenes; Pediococcus; Hybridization; Method; Strain specificity; Plate; Strain; Polymerase chain reaction; Streptococcaceae; Generation; DNA; Bacteria; Micrococcales; Technique
• ISSN: 0723-2020; 1618-0984
• DOI: 10.1078/0723-2020-00014

An improved subtraction hybridization technique was developed and evaluated. The hybridization is performed in a microplate with the subtractor-DNA immobilized in the plate while the probe-DNA is in solution. After hybridization the probe-specific DNA can easily be removed from the microwell and submitted to further analysis. This new technique has been successfully applied to generate several strain-specific PCR-primers for Lactococcus lactis subsp. lactis, Pediococcus spec., Saccharomyces spec. and Listeria monocytogenes.