DNA computing was proposed as a means of solving a class of intractable computational problems in which the computing time can grow exponentially with problem size (the 'NP-complete' or non-deterministic polynomial time complete problems). The principle of the technique has been demonstrated experimentally for a simple example of the hamiltonian path problem (in this case, finding an airline flight path between several cities, such that each city is visited only once). DNA computational approaches to the solution of other problems have also been investigated. One technique involves the immobilization and manipulation of combinatorial mixtures of DNA on a support. A set of DNA molecules encoding all candidate solutions to the computational problem of interest is synthesized and attached to the surface. Successive cycles of hybridization operations and exonuclease digestion are used to identify and eliminate those members of the set that are not solutions. Upon completion of all the multistep cycles, the solution to the computational problem is identified using a polymerase chain reaction to amplify the remaining molecules, which are then hybridized to an addressed array. The advantages of this approach are its scalability and potential to be automated (the use of solid-phase formats simplifies the complex repetitive chemical processes, as has been demonstrated in DNA and protein synthesis). Here we report the use of this method to solve a NP-complete problem. We consider a small example of the satisfiability problem (SAT), in which the values of a set of boolean variables satisfying certain logical constraints are determined.

A combination of scanning and imaging surface plasmon resonance (SPR) experiments is used to characterize DNA hybridization adsorption at gold surfaces and the subsequent immobilization of streptavidin. Single-stranded oligonucleotides are immobilized at gold surfaces, and the hybridization of biotinylated complements from solution is monitored with SPR. The subsequent attachment of streptavidin to the biotinylated complements provides a method of enhancing the SPR imaging signal produced as a result of the hybridization and leads to a 4-fold improvement in the hybridization detection limit of the SPR imaging apparatus. In situ scanning SPR experiments are used to measure a 60 ± 20% hybridization efficiency between immobilized single-stranded DNA and biotinylated complements. From the information provided by both the in situ imaging and scanning SPR experiments, an absolute surface coverage of immobilized single-stranded DNA is estimated to be ∼3 × 1012 molecules/cm2. The SPR signal resulting from hybridization onto immobilized probes is further amplified by the formation of streptavidin/DNA multilayers which grow by a combination of DNA hybridization and biotin−streptavidin binding. DNA/DNA multilayers without streptavidin are used as an additional method of amplifying the SPR signal.

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A new method for constructing oligonucleotide arrays on gold surfaces has been developed, and these arrays have been used in DNA hybridization experiments with in situ surface plasmon resonance (SPR) imaging detection. The detection technique was able to differentiate between single- and double-stranded DNA regions on the gold surface. The hybridization of both oligonucleotides and PCR-amplified DNA fragments was detectable, with the latter exhibiting slower hybridization kinetics. Temperature control of the in situ SPR cell was used to discriminate between perfectly matched duplexes and single-base-mismatched duplexes. The SPR detection technique requires no label on the DNA, but fluorescently labeled targets were also tested and detected by fluorescence imaging as an independent verification of the hybridization behavior of these DNA arrays. The in situ SPR imaging method for detection of DNA hybridization is expected to complement other existing methods for study of DNA interactions and might find future uses in mutation screening assays and DNA resequencing.

A strategy for DNA computing on surfaces using linked sets of 'DNA words' that are short oligonucleotides (16mers) is proposed. The 16mer words have the format 5'-FFFFvvvvvvvvFFFF-3' in which 4-8 bits of data are stored in 8 variable ('v') base locations, and the remaining fixed ('F') base locations are used as a word label. Using a template and map strategy, a set of 108 8mers each of which possesses at least a 4 base mismatch with the complements to all the other members of the set (4bm complements) are identified for use as a variable base sequence set. In addition, sets of 4 and 12 word labels of the form ABCD....DCBA that are respectively 8bm and 6bm complements with each other are identified. The 16mers are chosen to have a G/C content of 50% in order to make the thermodynamic stability of the perfectly matched hybridized DNA duplexes similar; a simple pairwise additive method is used to estimate the perfect match and mismatch hybridization thermodynamics. A series of preliminary experiments are presented that use small arrays of 16mers attached to chemically modified gold surfaces and fluorescently labeled complements to study the hybridization adsorption and enzymatic manipulation of the oligonucleotides.

The development of ultraminiaturized identification tags has applications in fields ranging from advanced biotechnology to security. This paper describes micrometer-sized glass barcodes containing a pattern of different fluorescent materials that are easily identified by using a UV lamp and an optical microscope. A model DNA hybridization assay using these ''microbarcodes'' is described. Rare earth-doped glasses were chosen because of their narrow emission bands, high quantum efficiencies, noninterference with common fluorescent labels, and inertness to most organic and aqueous solvents. These properties and the large number (>1 million) of possible combinations of these microbarcodes make them attractive for use in multiplexed bioassays and general encoding. Encoded bead bioassays are emerging as an attractive alternative to traditional slide-based microarrays because beadbased bioassays offer multiplexing of both probes and samples (the ''analyte''), and they have significantly fewer drawbacks related to mass transport-limited binding of analytes to the immobilized probes. Several approaches have been described for the fabrication of encoded beads: those in which the coding material is randomly distributed in the bead (1, 2) and those in which the coding material is present in a defined pattern on the bead (3). Because different patterns of the same coding materials (e.g., position and thickness of metal stripes on cylindrical particles) result in distinguishable beads (3), a larger number of uniquely encoded beads can be obtained relative to beads with randomly distributed coding materials (e.g., polymer beads infused with mixtures of quantum dots) (2).Current methods for fabricating encoded beads are limited in terms of either the number of possible codes or the compatibility of the beads with bioassays and fluorescence detection. The most widely used method for making encoded beads, infusing polymer microspheres with mixtures of fluorescent dyes in predefined ratios, is not well suited for the fabrication of large (Ͼ10 5 ) numbers of uniquely distinguishable beads. Trau and coworkers have used silica microspheres containing fluorescent dyes for encoding polymer beads by using split-pool methods, and have also described the formation of dye-doped concentric silica layers around core silica particles (4). There are only a limited number of spectrally well-resolved dyes that do not also interfere with commonly used biological labels. Moreover, measurements of intensities and their ratios are inherently difficult, which limits the number of levels at which a dye can be incorporated to give distinguishable beads. Mixtures of quantum dots embedded in polymer microspheres offer significant advantages over conventional fluorescent dyes because they are relatively more photostable and have narrow emission linewidths (2). However, quantum dots are made of toxic materials (e.g., CdS, CdSe, CdTe) (5), and difficulties distinguishing between codes based on different amounts of the same quantum dots are similar to those ...

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The application of resonance light scattering (RLS) particles for high-sensitivity detection of DNA hybridization on cDNA microarrays is demonstrated. Arrays composed of approximately 2000 human genes ("targets") were hybridized with colabeled (Cy3 and biotin) human lung cDNA probes at concentrations ranging from 8.3 ng/microL to 16.7 pg/microL. After hybridization, the arrays were imaged using a fluorescence scanner. The arrays were then treated with 80-nm-diameter gold RLS Particles coated with anti-biotin antibodies and imaged in a white light, CCD-based imaging system. At low probe concentrations, significantly more genes were detected by RLS compared to labeling by Cy3. For example, for hybridizations with a probe concentration of 83.3 pg/microL, approximately 1150 positive genes were detected using RLS compared to approximately 110 positive genes detected with Cy3. In a differential gene expression experiment using human lung and leukemia RNA samples, similar differential expression profiles were obtained for labeling by RLS and fluorescence technologies. The use of RLS Particles is particularly attractive for detection and identification of low-abundance mRNAs and for those applications in which the amount of sample is limited.