Biological lithography: development of a maskless microarray synthesizer for DNA chips

Cerrina, F.; Blattner, Fredrick R.; Huang, Wen; Hue, Yu; Green, R.; Singh‐Gasson, Sangeet; Sussman, Michael R.

doi:10.1016/s0167-9317(02)00566-x

Cited by 22 publications

(18 citation statements)

References 7 publications

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“…To identify genes under the control of MosR, cultures of both H37Rv and its isogenic ⌬mosR mutant were grown in vitro for DNA microarray analysis. A high-sensitivity oligonucleotide microarray platform was used in this experiment (7,37). Replicate microarray hybridizations were performed for at least two biological samples of both the wild-type and mutant strains and showed a high correlation level (r ϭ 0.9).…”

Section: Resultsmentioning

confidence: 99%

mosR , a Novel Transcriptional Regulator of Hypoxia and Virulence in Mycobacterium tuberculosis

et al. 2009

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Latent tuberculosis represents a high-risk burden for one-third of the world population. Previous analysis of murine tuberculosis identified a novel transcriptional regulator encoded by Rv0348 that could control the establishment of persistent tuberculosis. Disruption of the Rv0348 gene from the genome of the virulent H37Rv strain of Mycobacterium tuberculosis revealed a global impact on the transcriptional profiles of 163 genes, including induction of the mammalian cell entry (mce1) operon and the repression of a significant number of genes involved in hypoxia and starvation responses. Nonetheless, gel shift assays did not reveal direct binding between Rv0348 and a set of regulated promoters, suggesting an indirect regulatory role. However, when expressed in Mycobacterium smegmatis, the Rv0348 transcripts were significantly responsive to different levels of hypoxia and the encoded protein was shown to regulate genes involved in hypoxia [e.g., Rv3130c (tgs1)] and intracellular survival (e.g., mce1), among other genes. Interestingly, the colonization level of the ⌬mosR mutant strain was significantly lower than that of the wild-type strain of M. tuberculosis, suggesting its attenuation in the murine model of tuberculosis. Taken together, our analyses indicated that the Rv0348 gene encodes a novel transcriptional factor that regulates several operons involved in mycobacterial survival, especially during hypoxia; hence, we propose that Rv0348 be renamed mosR for regulator of mycobacterial operons of survival.

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Section: Resultsmentioning

confidence: 99%

mosR , a Novel Transcriptional Regulator of Hypoxia and Virulence in Mycobacterium tuberculosis

et al. 2009

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“…2 This technology utilizes a Digital Micromirror Device (DMD) produced by Texas Instruments, thus eliminating the time and cost usually associated with chrome masks and their alignment. 3 The frequency and distribution of errors in the sequences synthesized on chip is of primary importance. We have developed a simulation model to quantitatively calculate the locations and amounts of specific species: ideal sequence, mutants and single deletions/additions.…”

Section: Introductionmentioning

confidence: 99%

Biological lithography: Improvements in DNA synthesis methods

Kim

Rodesch

et al. 2004

Journal of Vacuum Science &Amp; Technology B: Microelectronics and Nanometer Structures Processing, Measurement, and Phenomena

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We have recently succeeded in synthesizing long oligonucleotides (90-mers) with high yield. This synthesis requires 360 virtual masks, and thus puts challenges on image placement and local contrast. We have updated our DNA synthesis modeling to Monte Carlo simulation from numerical approach. We also devised a method, called “Inverted Capping,” to remove sequence errors from edge scattering of light, which provides a large error reduction and the possibility of fabrication of higher resolutions. Finally, we have also implemented an image locking method to eliminate image drifts.

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“…Manual stages holding the reaction cell and slide assembly were used to position the cuts on the slide relative to the projected millichip image by observing their relative positions with the CCD camera assembly. Oligonucleotide probe synthesis was performed using DNA synthesis protocols previously described (Singh-Gasson et al, 1999;Cerrina et al, 2002;Nuwaysir et al, 2002) where the removal of the photo-labile protecting group 2-nitrophenylpropyloxycarbonyl was performed by exposure to broadband UVlight wavelengths of g, h, and i lines produced by a 350 Hg arc lamp (Newport) and nucleotide base attachment achieved using standard phosphoramidite chemistry (Fodor et al, 1991). After completion of microarray synthesis, the slide was broken into individual millichips by pressing a small-diameter precision drill bit (0.127-mm diameter) against the glass surface opposite to the slide cuts.…”

Section: Microarray Synthesismentioning

confidence: 99%

“…The MAS is an automated instrument that utilizes the Texas Instruments digital micromirror device (DMD; Sampsell, 1994) to perform photolithography and is uniquely capable of de novo synthesis of long (e.g. 60-mer) oligonucleotides on a glass surface with a density that exceeds 1 million or more probes patterned within a 2-cm squared glass surface and affords the capability of programming any desired probe sequence of reasonable length (Singh-Gasson et al, 1999;Cerrina et al, 2002;Nuwaysir et al, 2002).…”

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confidence: 99%

DNA Millichips as a Low-Cost Platform for Gene Expression Analysis

et al. 2012

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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.

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Biological lithography: development of a maskless microarray synthesizer for DNA chips

Cited by 22 publications

References 7 publications

mosR , a Novel Transcriptional Regulator of Hypoxia and Virulence in Mycobacterium tuberculosis

mosR , a Novel Transcriptional Regulator of Hypoxia and Virulence in Mycobacterium tuberculosis

Biological lithography: Improvements in DNA synthesis methods

DNA Millichips as a Low-Cost Platform for Gene Expression Analysis

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