Table 1.
Properties of microarray capture probes.
Figure 1.
Correlation of probe signal intensity and relative probe position.
Measured s/as-ratio of 115 probe pairs in logarithmic scale after hybridization of their respective specific target in relation to their probe position score.
Figure 2.
Position and nomenclature of the primers and probes within their target gene dfrA1.
The sense strand, forward primer, and probes in sense configuration (s) are displayed in light grey, whereas antisense stand, reverse primer, and probes in antisense configuration (as) are displayed in dark grey, respectively.
Figure 3.
Hybridization of products of four equimolar PCRs to seven probe pairs.
Signal intensities of the seven dfrA1 specific probe pairs (P1–P7) in sense and antisense configuration after hybridization of the dfrA1 PCR-product, which was amplified with four different primer concentrations.
Figure 4.
Hybridization of products of eight equimolar PCRs to probe pair 1.
Signal intensities of probe1_s and probe1_as after hybridization of the dfrA1 PCR-product, which was amplified with eight different primer concentrations.
Figure 5.
Hybridization of products of four asymmetric PCRs to seven probe pairs.
Signal intensities of probe pairs 1–7 after hybridization of the target dfrA1 which was amplified in four asymmetric PCRs containing different primer concentrations.
Figure 6.
Hybridization behavior of untreated PCR-product in comparison to PCR-product with decreased ssDNA content.
(A) gel picture of two samples: untreated dfrA1 PCR-product (lane 1) and purified and ExoI digested PCR-product (lane 2); (B) Signal intensities of probe pairs 1–7 after hybridization of untreated PCR-product generated with a primer concentration of 250 nM; (C) Signal intensities after hybridization of ExoI treated PCR-product. The digested sample was amplified under identical conditions as the control and adjusted to the same concentration after digestion.
Figure 7.
Hybridization behavior of PCR-products supplemented with ssDNA.
Signal intensities of probe pairs 1–7 after hybridization of the target dfrA1 without (A), with 10 nM (B), and 20 nM synthetic not-labeled ssDNA (C). PCR-products were generated with a primer concentration of 125 nM. The addition of synthetic sense strand led to a drastic change of the hybridization pattern by switching the binding preference of the target dfrA1 to the antisense configuration of each probe.
Figure 8.
Hybridization behavior of selectively labeled PCR-products supplemented with ssDNA.
Signal intensities of the seven dfrA1-specific probe pairs after hybridization of the target dfrA1 consisting of a labeled antisense and a not-labeled sense strand. The PCR-product was generated with a primer concentration of 125 nM and supplemented with 20 nM synthetic sense strand before hybridization.
Figure 9.
Model for the ssDNA induced hybridization of a PCR-product to microarray probes.
(A) PCR-product with an excess of one strand in solution over a microarray probe spot. (B) The ssDNA hybridizes to the probes and its dangling end can interact with the dsDNA. (C) Via a branch migration mechanism one strand of the dsDNA can be transferred to the former ssDNA. (D) When the strand is completely transferred, the laid off complementary strand can bind to a probe again and catalyze the hybridization of another dsDNA.
Figure 10.
Schematic formation of a double-stranded full-length PCR-product and abortion products of different lengths.
The abortion products of the sense strand lead to an excess of sense ssDNA in the vicinity of the forward primer binding site and those of the antisense strand in the vicinity of the reverse primer binding site, respectively. When both strands are formed to the same extend the equilibrium of sense and antisense strands lies in the middle of the product. The formation of one strand in excess leads to a shift of the equilibrium to one side.