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Nucleic Acids Research, 2001, Vol. 29, No. 19 e92
© 2001 Oxford University Press

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

Natalia E. Broude1,*, Karen Woodward2, Robert Cavallo2, Charles R. Cantor1,3 and David Englert4

1Center for Advanced Biotechnology and Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA, 2Packard BioChip Technologies, LLC Meriden, CT 06450, USA, 3Sequenom Inc., San Diego, CA 92121, USA and 4Packard BioScience Inc., Meriden, CT 06450, USA

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.

* To whom correspondence should be addressed at: Center for Advanced Biotechnology, 36 Cummington Street, Boston, MA 02215, USA. Tel: +1 617 353 8490; Fax: +1 617 353 8501; Email: nebroude{at}hotmail.com


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