Comparison of whole genome amplification methods for detecting pathogenic bacterial genomic DNA using microarray.
The genetic diagnosis of pathogenic agents using microarrays has the advantage of high-throughput detection, but a relatively large amount of DNA sample is required. To obtain a sufficient amount of DNA for molecular diagnoses, several whole genome amplification (WGA) methods have been proposed. In this study, using Francisella tularensis and Escherichia coli as models, we compared four WGA methods in terms of their efficiency of amplification of whole genomic DNA in order to identify the most suitable method for preparing DNA to be used for microarray analysis. It was possible to obtain more than 1.5 microg of products from 10 ng of F. tularensis and E. coli genomic DNA using four methods, but biases in the amplification of bacterial genes were least prominent in the multiple displacement amplification (MDA) or OmniPlex WGA. When the amplified DNAs were applied to microarray slides consisting of 32 different genes probes, DNAs amplified by Phi29 v2 of MDA and OmniPlex WGA showed high signal intensity as well as a high signal-to-noise ratio for all 32 genes. These results indicate that Phi29 v2 and OmniPlex WGA are useful methods for obtaining sufficient DNA from a limited amount of samples for the detection of microbes using microarrays.
Uda
Akihiko
A
Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo 162-8640, Japan. auda@nih.go.jp
Tanabayashi
Kiyoshi
K
Fujita
Osamu
O
Hotta
Akitoyo
A
Yamamoto
Yoshie
Y
Yamada
Akio
A
D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D005604:Francisella tularensis / Q000235:genetics / Q000302:isolation & purification; D016680:Genome, Bacterial; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction
D004269:DNA, Bacterial