BackgroundLight-directed in situ synthesis of DNA microarrays using computer-controlled projection from a digital micromirror device--maskless array synthesis (MAS)--has proved to be successful at both commercial and laboratory scales. The chemical synthetic cycle in MAS is quite similar to that of conventional solid-phase synthesis of oligonucleotides, but the complexity of microarrays and unique synthesis kinetics on the glass substrate require a careful tuning of parameters and unique modifications to the synthesis cycle to obtain optimal deprotection and phosphoramidite coupling. In addition, unintended deprotection due to scattering and diffraction introduce insertion errors that contribute significantly to the overall error rate.ResultsStepwise phosphoramidite coupling yields have been greatly improved and are now comparable to those obtained in solid phase synthesis of oligonucleotides. Extended chemical exposure in the synthesis of complex, long oligonucleotide arrays result in lower--but still high--final average yields which approach 99%. The new synthesis chemistry includes elimination of the standard oxidation until the final step, and improved coupling and light deprotection. Coupling Insertions due to stray light are the limiting factor in sequence quality for oligonucleotide synthesis for gene assembly. Diffraction and local flare are by far the largest contributors to loss of optical contrast.ConclusionsMaskless array synthesis is an efficient and versatile method for synthesizing high density arrays of long oligonucleotides for hybridization- and other molecular binding-based experiments. For applications requiring high sequence purity, such as gene assembly, diffraction and flare remain significant obstacles, but can be significantly reduced with straightforward experimental strategies.

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Our goal was to create a DNA chip that is as easy, convenient, and inexpensive as an agarose gel. For a first-generation solution, we describe a low-cost, easy-to-use de novo synthesis oligonucleotide microarray technology that draws on the inherent flexibility of the maskless array synthesizer for in situ synthesis of thousands of photolithographically produced oligonucleotides covalently attached to a microscope slide. The method involves physically subdividing the slide into 1 3 1 mm millichips that are hybridized to fluorescent RNA or DNA of biological origin, in a microfuge tube at an ordinary laboratory benchtop, rather than in dedicated hybridization chambers. Fluorescence intensity is then measured with a standard microscope rather than sophisticated DNA chip scanners. For proof of principle, we measured changes in the transcriptome of Arabidopsis (Arabidopsis thaliana) plants induced by growth in the presence of three major environmental abiotic stresses (temperature, light, and water status), in all possible combinations. Validation by comparison with quantitative reverse transcription PCR showed a high correlation coefficient and analysis of variance indicated a high technical reproducibility. These experiments demonstrate that low-cost DNA millichips can be made and reliably used at the benchtop in a normal laboratory setting, without assistance of core facilities containing costly specialized instrumentation.

This paper presents a detailed study on computer simulations of resist profiles obtained in x-ray lithography for exposures made either with synchrotron radiation or with an Al–Kα source. It is assumed, for purposes of the calculations, that the vacuum windows consist of kapton and that silicon is used as the mask material. The influence of edge shape and mask absorber thickness upon the resist structure is of special interest. The other parameters affecting resist profiles, such as Fresnel diffraction (especially in the case of semitransparent absorbers) and photoelectron range, are taken into consideration. In the case of the x-ray tube, the penumbral blur caused by the finite dimensions of the source spot leads to an additional deterioration of the edge sharpness. For the calculations, the intensity distribution over the spot area was assumed to be uniform (with Gaussian-shaped edges). The influence of the photoelectron range upon the resist profiles is calculated, using the simple depth-dose relationship of Gruen. The calculated resist profiles are compared with typical experimental results.

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This paper presents a detailed discussion of problems concerning mask heating, temperature distributions and resulting distortions of a boron-doped silicon mask which is exposed to intense x-ray radiation (from electron storage rings or plasma sources), having different spatial geometries and different distributions over time. For the calculations, all significant heat-loss mechanisms, e.g., radiation, thermal conduction in the mask, and heat transfer through an ambient gas, have been taken into consideration. The line-shaped synchrotron beam has a vertical Gaussian intensity distribution, with nearly time-constant radiation power. The influences of different scan modes on the temperature rise are taken into account. In the case where helium is used as the coolant gas, the temperature rise remains within acceptable limits, even at the highest possible radiation power. The plasma source emits extremely short, high power x-ray pulses having a homogeneous spatial distribution. The short pulse duration leads to a considerable temperature rise in the mask, which would introduce appreciable distortions. However, the homogeneity of the exposure combined with the stress in the mask membrane can suppress this effect.

Resolution in x-ray lithography is influenced mainly by the exposure geometry, the resist's behavior, and by the Fresnel diffraction as well. The range of photoelectrons created by the soft x rays, which can also effect the resolution, has been investigated theoretically and experimentally. The effective range remains below 20 nm depending on the resist material and on the exposure wavelength. As the photoelectrons do not limit the resolution in synchrotron lithography under practical conditions (proximity gap;>20 ,urn), this effect is not considered explicitly in the x~ray lithography simulator XMAS which enables the three-dimensional simulation of resist profiles. The application of XMAS to synchrotron lithography and the related one-layer resist systems reveals a Fresnel-limited resolution somewhere between 0.1 and 0.2 ,urn, depending on the actual resist parameters.