A versatile maskless microscope projection photolithography system and its application in light-directed fabrication of DNA microarrays

We present a maskless microscope projection lithography system (MPLS), in which photomasks have been replaced by a Digital Micromirror Device type spatial light modulator (DMD, Texas Instruments). Employing video projector technology high resolution patterns, designed as bitmap images on the computer, are displayed using a micromirror array consisting of about 786000 tiny individually addressable tilting mirrors. The DMD, which is located in the image plane of an infinity corrected microscope, is projected onto a substrate placed in the focal plane of the microscope objective. With a 5x(0.25 NA) Fluar microscope objective, a fivefold reduction of the image to a total size of 9 mm2 and a minimum feature size of 3.5 microns is achieved. Our system can be used in the visible range as well as in the near UV (with a light intensity of up to 76 mW/cm2 around the 365 nm Hg-line). We developed an inexpensive and simple method to enable exact focusing and controlling of the image quality of the projected patterns. Our MPLS has originally been designed for the light-directed in situ synthesis of DNA microarrays. One requirement is a high UV intensity to keep the fabrication process reasonably short. Another demand is a sufficient contrast ratio over small distances (of about 5 microns). This is necessary to achieve a high density of features (i.e. separated sites on the substrate at which different DNA sequences are synthesized in parallel fashion) while at the same time the number of stray light induced DNA sequence errors is kept reasonably small. We demonstrate the performance of the apparatus in light-directed DNA chip synthesis and discuss its advantages and limitations.Comment: 12 pages, 9 figures, journal articl

Position dependent mismatch discrimination on DNA microarrays – experiments and model

<p>Abstract</p> <p>Background</p> <p>The propensity of oligonucleotide strands to form stable duplexes with complementary sequences is fundamental to a variety of biological and biotechnological processes as various as microRNA signalling, microarray hybridization and PCR. Yet our understanding of oligonucleotide hybridization, in particular in presence of surfaces, is rather limited. Here we use oligonucleotide microarrays made in-house by optically controlled DNA synthesis to produce probe sets comprising all possible single base mismatches and base bulges for each of 20 sequence motifs under study.</p> <p>Results</p> <p>We observe that mismatch discrimination is mostly determined by the defect position (relative to the duplex ends) as well as by the sequence context. We investigate the thermodynamics of the oligonucleotide duplexes on the basis of double-ended molecular zipper. Theoretical predictions of defect positional influence as well as long range sequence influence agree well with the experimental results.</p> <p>Conclusion</p> <p>Molecular zipping at thermodynamic equilibrium explains the binding affinity of mismatched DNA duplexes on microarrays well. The position dependent nearest neighbor model (PDNN) can be inferred from it. Quantitative understanding of microarray experiments from first principles is in reach.</p

Summarizing activity limitations in children with chronic illnesses living in the community: a measurement study of scales using supplemented interRAI items

<p>Abstract</p> <p>Background</p> <p>To test the validity and reliability of scales intended to measure activity limitations faced by children with chronic illnesses living in the community. The scales were based on information provided by caregivers to service program personnel almost exclusively trained as social workers. The items used to measure activity limitations were interRAI items supplemented so that they were more applicable to activity limitations in children with chronic illnesses. In addition, these analyses may shed light on the possibility of gathering functional information that can span the life course as well as spanning different care settings.</p> <p>Methods</p> <p>Analyses included testing the internal consistency, predictive, concurrent, discriminant and construct validity of two activity limitation scales. The scales were developed using assessment data gathered in the United States of America (USA) from over 2,700 assessments of children aged 4 to 20 receiving Medicaid Early and Periodic Screening, Diagnostic and Treatment (EPSDT) services, specifically Personal Care Services to assist children in overcoming activity limitations. The Medicaid program in the USA pays for health care services provided to children in low-income households. Data were collected in a single, large state in the southwestern USA in late 2008 and early 2009. A similar sample of children was assessed in 2010, and the analyses were replicated using this sample.</p> <p>Results</p> <p>The two scales exhibited excellent internal consistency. Evidence on the concurrent, predictive, discriminant, and construct validity of the proposed scales was strong. Quite importantly, scale scores were not correlated with (confounded with) a child's developmental stage or age. The results for these scales and items were consistent across the two independent samples.</p> <p>Conclusions</p> <p>Unpaid caregivers, usually parents, can provide assessors lacking either medical or nursing training with reliable and valid information on the activity limitations of children. One can summarize these data in scales that are both internally consistent and valid. Researchers and clinicians can use supplemented interRAI items to provide guidance for professionals and programs serving children, as well as older persons. This research emphasizes the importance of developing medical information systems that allow one to integrate information not only across care settings but also across an individual's life course.</p

Optical Study of DNA Surface Hybridization Reveals DNA Surface Density as a Key Parameter for Microarray Hybridization Kinetics

We investigate the kinetics of DNA hybridization reactions on glass substrates, where one 22 mer strand (bound-DNA) is immobilized via phenylene-diisothiocyanate linker molecule on the substrate, the dye-labeled (Cy3) complementary strand (free-DNA) is in solution in a reaction chamber. We use total internal reflection fluorescence for surface detection of hybridization. As a new feature we perform a simultaneous real-time measurement of the change of free-DNA concentration in bulk parallel to the total internal reflection fluorescence measurement. We observe that the free-DNA concentration decreases considerably during hybridization. We show how the standard Langmuir kinetics needs to be extended to take into account the change in bulk concentration and explain our experimental results. Connecting both measurements we can estimate the surface density of accessible, immobilized bound-DNA. We discuss the implications with respect to DNA microarray detection