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User applications related to surface binding/immobilization and microarrays

Modified oligos for microarrays

Dr. Ana García-Sacristán, part of Dr. Carlos Brione's group, in the Department of Molecular Evolution at the Centro de Astrobiología (CAB), uses modified DNA oligonucleotides synthesized by IBA for optimizing new applications of DNA microarrays-based methodologies. "In particular, we are currently taking advance of the performance of IBA technology for the synthesis of DNA oligonucleotides modified with a "C6" amino linker [NH2(CH2)6] at their 5´end. We immobilize such modified oligos onto microarray slides with a super-epoxy-coated surface. The amino-modified 5' end of the oligos is followed by a 15 nt-long DNA spacer and the specific sequence complementary to the region of interest. We are currently using our DNA microarrays for the structural characterization of native viral genomic RNA previously labeled with fluorescent dyes."

Oligonucleotides grafted to charged surfaces

24mer and 48mer oligonucleotides, Cy3-labeled at the 3’-end and modified with a thiol linker at the 5’- end, were custom synthesized by IBA. The thiol linker was used to chemically graft the strands to a gold surface (S-Au bond), including gold electrodes. Firstly, single stranded oligonucleotides were adsorbed onto the surface and used for measurements. The layers were then hybridized with nucleic acids of complementary sequence, so that measurements could be repeated in the double stranded conformation.
Measurements were performed by applying a low frequency (0.2 Hz), square wave potential modulation to the gold electrodes. Simultaneously, the fluorescence emitted from dye labels attached to the DNAs’ distal ends was monitored. The intrinsically negatively charged DNA is either repelled from the negatively charged, or attracted towards the positively charged electrode surface (Figure 1).
With this experimental setup, Rant et al. investigated the influence of the applied field strength or effects of the surrounding electrolyte solution and found that single- and double-stranded DNA exhibit differences because of their dissimilar flexibility. The findings arethought to hold true for charged, linear macromolecules which are exposed to high field gradients at interfaces.

 

 

 

Figure 1: Electrically switchable DNA layers. Alternating potentials are applied to the gold substrate. The negatively charged DNA is repelled from negatively, or attracted to positively charged electrodes, respectively. The dashed rays indicate the intensity of the fluorescence emission, which is quenched if the dye approaches the metal surface.

Literature:

Ulrich Rant, Kenji Arinaga, Shozo Fujita, Naoki Yokoyama, Gerhard Abstreiter and Marc Tornow (2006)
Electrical manipulation of oligonucleotides grafted to charged surfaces.
Organic & Biomolecular Chemistry 4: 3448–3455. DOI: 10.1039/B605712H – Reproduced by permission of The Royal Society of Chemistry.

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Appl Note_chargedsurface.pdf Appl Note_chargedsurface.pdf (323.6 kB)
DNA binding to a PNA covered surface

Cattani-Scholz et al. describe surface covering with PNA (peptide nucleic acid), which is bound via a linker1 to a phosphonate carbon chain2, which is in turn bound to a siliconoxide-coated silicon surface (Fig. 1). DNA strands, custom manufactured by IBA, can bind to this PNA covered surface. In contrast to DNA, PNA does not have an anionic phosphate backbone, and is therefore uncharged at neutral pH, leading to lack of repulsion and enhanced hybridization efficiency.
Cattani-Scholz et al. also show that their functionalization protocol can be translated from planar Si substrates to Si nanowire sensor devices. They employ these nanowire sensors to measure the change in surface potential upon PNA/DNA hybridization via the field effect.

Figure 1: Schematic illustration of the stepwise functionalization of the SiO2-terminated Si surface. PNA is coupled to a monolayer of 11-hydroxyundecylphosphonate using the heterobifunctional linker1 (1). Following attachment via the maleimido moiety, single stranded PNA serves as a receptor for hybridization with target DNA (synthesized by IBA). Reprinted with permission from Cattani-Scholz et al., 2008. Copyright 2008 American Chemical Society.

13-maleimidopropionic-acid-N-hydroxysuccinimide
211- hydroxyundecylphosphonate

Literature:

Anna Cattani-Scholz, Daniel Pedone, Manish Dubey, Stefan Neppl, Bert Nickel, Peter Feulner, Jeffrey Schwartz, Gerhard Abstreiter, and Marc Tornow (2008)
Organophosphonate-Based PNA Functionalization of Silicon Nanowires for Label-Free DNA Detection.
ACS nano 2(8):1653–1660

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Appl Note_DNA PNA.pdf Appl Note_DNA PNA.pdf (357.6 kB)
Point mutations in DNA and RNA duplex formation (microarray)

Cy3-labeled target oligonucleotides (DNA and RNA, Figure 1) were synthesized by IBA. Complementary probe sequence motifs with point mutations were spotted on a microarray so that hybridization of the target oligonucleotides could be measured. The influence of the different kinds of point mutations (mismatch, insertion, deletion) and the different positions of the defect on the oligonucleotide duplex binding affinity were compared.

Figure 1: Design of the experiment. A comprehensive set of point-mutated probes is derived from a common probe sequence motif which is complementary to the target sequence. Probe sequences are shown for the first two defect positions only. To enhance quantitative analysis probe sequences are arranged on the microarray as a compact feature block. Reproduced by permission of BioMed Central.

Naiser et al. found that DNA microarrays can resolve even subtle changes in hybridization affinity for simple target mixtures.

Literature

Thomas Naiser, Oliver Ehler, Jona Kayser, Timo Mai, Wolfgang Michel and Albrecht Ott (2008)
Impact of point-mutations on the hybridization affinity of surface-bound DNA/DNA and RNA/DNA oligonucleotide-duplexes: Comparison of single base mismatches and base bulges.
BMC Biotechnology 8:48.

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Appl Note_duplex formation.pdf Appl Note_duplex formation.pdf (772.3 kB)