DNA microarrays with stem-loop DNA probes: preparation and applications

We have developed DNA microarrays containing stem-loop DNA probes with shor t single-stranded overhangs immobilized on a Packard HydroGel chip, a 3-dim ensional porous gel substrate. Microarrays were fabricated by Immobilizing self-complementary single-stranded oligonucleotides, which adopt a partiall y duplex structure upon denaturing and reannealing. Hybridization of single -stranded DNA targets to such arrays Is enhanced by contiguous stacking Int eractions with stem-loop probes and Is highly sequence specific. Subsequent enzymatic ligation of the targets to the probes followed by stringent wash ing further enhances the mismatched base discrimination. We demonstrate her e that these microarrays provide excellent specificity with signal-to-backg round ratios of from 10- to 300-fold. In a comparative study, we demonstrat ed that HydroGel arrays display 10-30 times higher hybridization signals th an some solid surface DNA microarrays. Using Sanger sequencing reactions, w e have also developed a method for preparing nested 3 ' -deletion sets from a target and evaluated the use of stem-loop DNA arrays for detecting p53 m utations In the deletion set. The stem-loop DNA array format Is simple, rob ust and flexible In design, thus It Is potentially useful In various DNA di agnostic tests.