Multiple displacement amplification as an aid in checkerboard DNA-DNA hybridization.

OBJECTIVE

The study aimed to determine if multiple displacement amplification could be used to provide abundant target DNA and DNA probes for checkerboard DNA-DNA hybridization.

METHODS

Multiple displacement amplification was used to amplify 1 and 10 ng DNA from 16 individual bacterial species, DNA from single colonies, from a mixture of 20 bacterial species and oral biofilm samples, such as supragingival plaque, subgingival plaque, buccal swab and root canal samples. Samples in reaction buffer were heat-denatured at 95 degrees C for 3 min and cooled to 4 degrees C. Phi29 DNA polymerase was added and the mixture was incubated at 30 degrees C for 16-18 h. The quantity of the product was evaluated by the Picogreen assay. The amplified material was labeled with digoxigenin. The probes were compared with probes obtained from unamplified DNA using checkerboard DNA-DNA hybridization. Both amplified DNA and unamplified DNA were used as targets on the membrane. Amplified oral biofilm samples were compared to unamplified samples using checkerboard DNA-DNA hybridization.

RESULTS

The DNA yield ranged from 4 to 11 microg. DNA-DNA hybridization showed that the amplified genome of each species used either as target or as probe provided signals equivalent to controls and that amplification of a mixture of species provided signals comparable to those provided by the unamplified source mixture. Amplified oral biofilm samples exhibited comparable proportions of bacterial DNA when compared to the original unamplified samples.

CONCLUSIONS

The multiple displacement amplification technique is a simple and reliable method to uniformly amplify DNA for use in checkerboard DNA-DNA hybridization. It is also a useful tool in the amplification of clinical samples.