Our platform

We have developed an extremely flexible synthesis technology that enables us to manufacture custom microarray slides and oligonucleotide libraries at a very competitive price, with no upfront customization costs. Our synthesis technology begins with the synthesis of DNA oligonucleotide libraries on microarray slides. These oligos can be then cleaved and/or modified for countless downstream applications. We have combined the flexibility of digital photolithography to the robustness of the phosphoramide oligonucleotide synthesis chemistry. This enables us to synthesize up to 500,000 spots on substrates the size of a microscope slide at a very competitive cost. Since we are using a mask-less approach, our technology is extremely flexible, making possible to offer custom synthesis with no setup cost or minimum order. Read below to learn more about different technological features of our platform.

I. Flexible in-situ synthesis

Proprietary light directed oligonucleotide synthesis

We do not spot presynthesized oligonucleotides. We directly synthesize them on a microscope slide. Our synthesis is using the robust phosphoramidite chemistry refined since the 1970's for commercial oligonucleotide synthesis. In standard oligonucleotide synthesis, the terminal acid-labile protective group of a growing oligomer is removed by a treatment with a strong acid.

The only modification we have introduced in our process is the usage of a photo-generated acid. At a given synthesis cycle, each spot that needs to be deprotected is exposed to light in the presence of a photogenerated acid precursor, leading to local acid production and cleavage of the terminal protective group. Only the deprotected spots will be able to react with a new monomer. By so doing, we can synthesize tens of thousands oligonucleotides in parallel on the same microarray substrate.

The advantage of using the phosphoramidite chemistry is that we can synthesize oligonucleotides at a reduced cost compared to other chemistries while maintaining our stepwise yield well above 99%.

Here is an animation showing several synthesis cycles:

Image description

Ultra-flexible digital photolithography

To expose the oligonucleotide spots to light during the deprotection step of the synthesis cycle, we do not use any physical mask but instead we use a digital micromirror device (DMD) made of several hundred of thousands individually addressable mirrors. At each deprotection step, a computer simply generates an image to be projected on the microarray surface. Using this digital photolithography approach, we can synthesize any custom microarray with no upfront design cost or minimal order size.

High quality glass substrates

We synthesize our microarrays on high quality microscope slides. For uniform hybridization and enhanced scanner image quality, we use only low-background glass slides polished to atomic flatness (±20 angstroms). Their precise physical dimension (25 ± 0.2 mm x 76 ± 0.3 mm x 0.940 mm ± 0.025 mm) is compatible with all major brands of slide microarray scanners.

II. Uniform spot morphology

Highly uniform

The combination of the robust phosphoramidite oligonucleotide chemistry and a very high contrast ratio light projector enables us to routinely achieve very high spot uniformity. With our technology, "doughnuts" spots belong in the past.

In the following experiment, spots on a microarray (45-mer probe) were hybridized with a synthetic Cy3-labeled target. The upper panel shows spots as viewed in GenePix Pro 6.0 software, as well as a cross-section of a spot (5 microns resolution). The lower panels show 3-dimensional views of the same spots. Our in situ synthesis technology produces highly uniform spot morphology with very sharp edges.

Spot uniformity

High signal-to-noise ratio

This translates into high quality microarray images with very high signal to noise ratio using a scanner's full dynamic range. Spots are evenly spaced in both dimensions and accurately fit the software's microarray analysis grid for easy quantification.

Below is a an image from a typical experiment where RNA extracted from a bacterial mutant strain and its wild type counterpart were fluorescently labeled with Alexa fluors (555 and 647, respectively) and cohybidized on an array of 45-mer probes designed against the transcriptome of this bacteria. Click here to open a high-resolution full size image in a new window (12MB).

Dual color microarray image

III. Excellent reproducibility

Experimental design

In order to measure the reproducibility of our microarrays in a real experimental setup, we have designed a yeast 30K microarray containing 6540 probe sets that survey 6331 unique transcripts. Each probe set contains 4 identical probe replicates that are randomly distributed across the array. We have synthesized nine yeast microarrays (3 batches of 3 slides). Yeast was grown on two different sources of carbon and total RNA were extracted, amplified and labeled with two different fluorophores. A single master hybridization mix was prepared and hybridized to the 9 slides.

For the coefficient of variation (CV) analysis, the ratio (F635/F532) of scaled and background subtracted median signal was used. Spots with signal that did not exceed twice the background in both channels were considered "not present" and eliminated from further analysis. Probe sets qualified for CV analysis if all 4 identical probe replicates within an array had signal, in at least one channel, which exceeded twice the background level in the appropriate channel.

Intra-array coefficient of variation

For the coefficient of variation (CV) analysis, the ratio (F635/F532) of scaled and background subtracted median signal was used. Spots with signal that did not exceed twice the background in both channels were considered "not present" and eliminated from further analysis. Probe sets qualified for CV analysis if all 4 identical probe replicates within an array had signal, in at least one channel, which exceeded twice the background level in the appropriate channel.

Intra-array coefficient of variation

Green: Batch 1, slides 1 to 3; Yellow: Batch 2, slides 1 to 3; Red: Batch 3, Slides 1 to 3.

Inter-array coefficient of variation

Inter-array intra-batch CVs were calculated for each probe if all three probes qualified (signal in at least one channel exceeded twice the background level in the appropriate channel). As seen in the graph below, our inter-array mean CVs are less than 12% (μ = 9.3 ± 2.3). Importantly, no inter-array normalization was performed prior to calculating inter-array CV. These data demonstrate that high signal uniformity is maintained across arrays synthesized contemporaneously.

Inter-array coefficient of variation

Inter-batch coefficient of variation

Inter-array CV was calculated for each probeset across 9 arrays from 3 independent batches of arrays. Inter-batch CVs were calculated using a variable number (≥4, ≥7, or all 9) of qualifying probesets (see above). As seen in the figure below, inter-batch CVs were less than 10% (μ = 8.8 ± 0.8) and independent of the number of qualifying probe sets. Importantly, no inter-array or inter-batch normalizations were performed. These data show that high signal constancy is preserved across arrays that were manufactured in different batches.

Inter-batch coefficient of variation

IV. Cross-platform compatibility

Standard slide format

We synthesize our microarrays on high quality microscope slides. For uniform hybridization and enhanced scanner image quality, we use only low-background glass slides polished to atomic flatness (±20 angstroms). Their precise physical dimension (25 ± 0.2 mm x 76 ± 0.3 mm x 0.940 mm ± 0.025 mm) is compatible with all major brands of slide microarray scanners.

Hybridization & washing

We have successfully tested our slides in various systems, from simple hybridization under a coverslip in a humid chamber to sophisticated hybridization and washing stations. Our slides are even compatible with Agilent hybridization cassettes and gasket slides for increased versatility.

Scanning

Our slides can be read by any scanner accepting microscope slides, including autoloader scanners. Slides have a printed label with a barcode for easy tracking.

V. Sensitive & specific probe design

Probe design algorithm

Our proprietary probe design software searches for specific probes at the genomic level. The probe candidate is compared to all other expressed sequences from the same organism and the thermodynamic parameters (free energy and melting temperature) are computed for all possible hybridizations between the probe and perfect or non-perfect complementary sequences. If all of these values fall below a predetermined threshold, the probe is considered to be specific to its target. Probes are also selected to be unable to fold into stable secondary structures that may interfere with hybridization. Any probes with low sequence complexity or long stretches of the same base are rejected.

Catalog microarray probe design

In terms of sensitivity, and specificity, the optimal size for an oligonucleotide grown directly on a microarray and used for gene expression analysis is between 50 and 60 bases (Kane 2000, Hughes 2001). However, the 10 nucleotides closest to the chip's surface are not likely involved in hybridization due to steric interference (Shchepinov 1997). There is no reason, therefore, to consider these nucleotides during the design process and especially during the specificity computation, as long as spacer sequence of sufficient length is inserted between the target sequence and the chip surface during fabrication.

Accordingly, we have chosen to design probes with a size comprised between 45 and 47 nucleotides. By using a range of probe lengths, our probe design algorithm can fit a narrower melting temperature (Tm) range in order to achieve better hybridization uniformity. Our probes are synthesized on top of a spacer arm to get them away from the glass substrate. Our spacer has a length equivalent to a 15-mer sequence, which is longer than the 10 nucleotides recommended by Shchepinov et al.

Eukaryotic messenger RNAs are polyadenylated and since this feature is often used to anchor reverse transcription during probe labeling, we have limited the search space for probes to the last 1500 nucleotides of the input sequences. This limit is to prevent picking probes in a region that would eventually not be reverse transcribed in suboptimum experimental conditions. The input sequence is searched in a 3' to 5' direction to give preference to potential probe sequences that are located as closely as possible to the messenger's 3' end. For archaea and bacteria where the mRNAs are not polyadenylated, the reverse transcription is usually primed with short random primers. This will lead to a better representation of the 5' end of the mRNAs into the cDNA population. Thus, the input sequence is searched in a 5' to 3' direction to preferentially pick probes close to the messenger's 5' end.

References:

Hughes, T.R., Mao, M., Jones, A.R., Burchard, J., Marton, M.J., Shannon, K.W., Lefkowitz, S.M., Ziman, M., Schelter, J.M., Meyer, M.R. et al. (2001) Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nat Biotechnol, 19, 342-347.

Kane, M.D., Jatkoe, T.A., Stumpf, C.R., Lu, J., Thomas, J.D. and Madore, S.J. (2000) Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays. Nucleic Acids Res, 28, 4552-4557.

Shchepinov, M.S., Case-Green, S.C. and Southern, E.M. (1997) Steric factors influencing hybridisation of nucleic acids to oligonucleotide arrays. Nucleic Acids Res, 25, 1155-1161.