Quantitative detection of microbial genes by using DNA microarrays

Abstract

To quantify target genes in biological samples using DNA microarrays, we employed reference DNA to normalize variations in spot size and hybridization. This method was tested using nitrate reductase (nirS), naphthalene dioxygenase (nahA), and Escherichia coli O157 O-antigen biosynthesis genes as model genes and lambda DNA as the reference DNA. We observed a good linearity between the log signal ratio and log DNA concentration ratio at DNA concentrations above the method's detection limit, which was approximately 10 pg. This approach for designing quantitative microarrays and the inferred equation from this study provide a simple and convenient way to estimate the target gene concentration from the hybridization signal ratio.

FIG. 1.

(a) Schematic diagram showing microarray design and hybridization procedure. (b) Representative scanning image of microarray. A sample containing 10 ng of each model gene was used for the microarray hybridization. Pseudo-colors indicate the ratio (R) between Cy3 channel and Cy5 channel (green, R > 1; yellow, R = 1; red, R < 1).

FIG. 2.

Relationship between hybridization signal ratios and weight fraction of model genes, nirS (a), nahA (b), and E. coli O157 O-antigen biosynthesis gene (c). The solid and dotted lines indicate the regression curve and 95% prediction interval, respectively. Error bars indicate SDs.

FIG. 3.

Relationship between estimated model gene concentration and actual concentration of model genes, nirS (a), nahA (b), and E. coli O157 O-antigen biosynthesis gene (c). Closed circle, calculated by log (R′) = α log (f_x/f_λ) + β, in this study; open circle, estimation by hybridization kinetics. The solid line indicates the regression curve, and the diagonal dotted line indicates a 1:1 relationship. Error bars indicate standard deviations. No error bars are shown when smaller than symbols.