Rapid Exonuclease Digestion of PCR-Amplified Targets for Improved Microarray Hybridization

Hybridization of double-stranded DNA with nucleic acid probes is hampered by competition between the complementary nontarget strand and the probe (1). This competition is stronger with surface-bound probes that expose a target strand with a long dangling end toward the media(2)(3). This situation is more problematic in infectious disease diagnostics, which requires high sensitivity, sometimes as low as 1 genome copy. Microarray integration into microfluidic systems results in increased speed and sensitivity while allowing automation of the whole hybridization process(4) and could lead to the development of point-of-care diagnostic devices. Actual limitations of diagnostic microarrays include the design of ultrasensitive capture probes that are highly specific and discriminant. Some techniques have been developed to produce single-stranded DNA targets for sequencing and hybridization, but these methods may necessitate additional steps or modification of the target strand or have poor sensitivities(5)(6)(7)(8). Degradation of the complementary strand can alleviate competition problems and allow more flexible probe design for the target strand. Therefore, we developed a simple 5-min method for rapid single-step selective digestion of the complementary strand with lambda exonuclease, which leads to increased hybridization signals and improved differentiation of single-nucleotide polymorphisms (SNPs) on DNA microarrays. Cy3-labeled primers were used to generate the target strands, and phosphorylated primers were used to generate the complementary strands. Nonphosphorylated primers were also used to verify the protection provided by the Cy3-labeled primers. All oligonucleotides were purchased from Integrated DNA Technologies. Purified genomic DNA (1 ng) from Neisseria meningitidis (ATCC-13077), Listeria monocytogenes (CCRI-4862), and Candida krusei (ATCC-28870) were PCR-amplified with a PTC-200 thermocycler (Bio-Rad Laboratories; 1 min at 94 °C, then 40 cycles of 1 s at 95 °C for the denaturation step, 10 s at 60 °C for the annealing step, and 20 s at …