Journal:

Article Title: Signal amplification by rolling circle amplification on DNA microarrays

doi:

Figure Lengend Snippet: Metrics of RCA on glass arrays. (A) Schematic representation of RCA metrics. The accessibility of surface-immobilized primers was measured by hybridizing a Cy5-labeled decorator to primer 1 immobilized in a dilution series on microarrays. Hybridization of RCA circles to the immobilized primers was measured using a Cy5-labeled oligonucleotide probe complementary to circle 1 (which hybridizes to a different sequence of circle from the primer 1 annealing site). Accessibility of primed circles for initiation by a polymerase was measured by T7 Sequenase incorporation of a single Cy5-ddGTP nucleotide using circle 1 as the template. (B) Primer accessibility on planar microarrays. Fluorescence intensities of hybridization of the decorator probe (filled squares) and the RCA circle (filled circles) are shown. The efficiency of polymerase initiation was measured by single base extension (open circles). Fluorescence signal intensities are plotted against each concentration of primer deposited. Average background was subtracted from each data point. Error bars indicate the standard deviations from the means of two spots from three different arrays. (C) Comparison of RCA amplification and hybridization sensitivities on glass arrays. Microarrays containing a dilution series of immobilized primer 1 were hybridized with circle 1 and RCA amplification was performed using native T7 DNA polymerase. After RCA signal amplification, slides were washed as described and scanned using a ScanArray 5000 laser scanner and analyzed with the QuantArray software package (GSI Lumonics). The figure shows a plot of the average pixel intensity at each spot by RCA (filled circles) and hybridization (filled squares) after subtraction of the average background from each point. Error bars indicate the standard deviations from the means of 10 arrays and two spots per array. (D) Dynamic range of RCA signal amplification. The scanner settings were optimized to determine signal intensities at the lower (left) or higher (right) primer concentration ranges shown in (C).

Article Snippet: Images were analyzed with the QuantArray Image Analysis software package (GSI Lumonics) using the fixed circle method.

Techniques: Labeling, Hybridization, Sequencing, Fluorescence, Concentration Assay, Amplification, Software