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Use of a nonradioactive genetic probe identified, synthesized, and labeled in the polymerase chain reaction
Author(s) -
Preus Hans R.,
Russell Donald T.
Publication year - 1994
Publication title -
european journal of oral sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.802
H-Index - 93
eISSN - 1600-0722
pISSN - 0909-8836
DOI - 10.1111/j.1600-0722.1994.tb01173.x
Subject(s) - ethidium bromide , biology , polymerase chain reaction , primer (cosmetics) , microbiology and biotechnology , genomic dna , agarose gel electrophoresis , actinobacillus , dna , agarose , hybridization probe , molecular probe , genetics , bacteria , gene , chemistry , organic chemistry
This study introduces a strategy to identify and produce sequences useful as genetic markers, or native genetic probes for DNA‐DNA hybridization in bacterial strains where the genetics is not well described. Actinobacillus actinomycetemcomitans (A.a .) was used as an example. Fifty ng genomic DNA from A.a . ATCC 33384 and Haemophilus aphrophilus ATCC 33389 was amplified in a thermocycler using a single 10‐mer primer. The PCR products were separated by electrophoresis on a 1% submarine agarose gel containing ethidium bromide and visualized by UV illumination, and the strain‐specific amplitypes were compared. DNA from two bands, 0.9 and 4 kb, unique for the A.a . strain, was cut out, amplified under high stringency with the same primer and labeled by replacing 33.3 μM dTTP with digoxigenin‐labeled dUTP in the reaction mixture. The labeled probe was then repeatedly used for hybridization to DNA from various A.a., H. aphrophilus , and other bacterial strains of the Pasteurellaceae family. The results showed that the 0.9‐kb probe detected all A.a . tested, and distinguished it from other closely related bacterial species. We conclude that the described strategy is useful for identifying and selecting genetic sequences useful as genetic markers in A.a .