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

AUTOR(ES)

Broude, Natalia E.

FONTE

Oxford University Press

RESUMO

We have developed DNA microarrays containing stem–loop DNA probes with short single-stranded overhangs immobilized on a Packard HydroGel chip, a 3-dimensional porous gel substrate. Microarrays were fabricated by immobilizing self-complementary single-stranded oligonucleotides, which adopt a partially duplex structure upon denaturing and re-annealing. Hybridization of single-stranded DNA targets to such arrays is enhanced by contiguous stacking interactions with stem–loop probes and is highly sequence specific. Subsequent enzymatic ligation of the targets to the probes followed by stringent washing further enhances the mismatched base discrimination. We demonstrate here that these microarrays provide excellent specificity with signal-to-background ratios of from 10- to 300-fold. In a comparative study, we demonstrated that HydroGel arrays display 10–30 times higher hybridization signals than some solid surface DNA microarrays. Using Sanger sequencing reactions, we 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 mutations in the deletion set. The stem–loop DNA array format is simple, robust and flexible in design, thus it is potentially useful in various DNA diagnostic tests.

Documentos Relacionados

• Investigation of the multiple anchors approach in oligonucleotide microarray preparation using linear and stem–loop structured probes
• DNA stem-loop structures in oligopurine-oligopyrimidine triplexes.
• DNA stem-loop structures bind poorly to histone octamer cores.
• RNA Destabilization by the Granulocyte Colony-Stimulating Factor Stem-Loop Destabilizing Element Involves a Single Stem-Loop That Promotes Deadenylation
• Discovering common stem–loop motifs in unaligned RNA sequences