DNA arrays fabrication

A typical DNA array fabrication and application process involves three major steps. First, nucleic acids (the capture sequences or probes) are immobilized at discrete positions on surface activated substrates. Secondly, the resulting array is hybridized with a complex mixture of fluorescently labelled nucleic acids (the target), and thirdly subsequent to hybridization, the fluorescent markers are detected using a high-resolution scanning laser that quantifies the interaction. This chapter focuses on the first of these processes and provides the reader with an overview of substrates, surface activation methods and dehvery systems available for nucleic acid immobilization. 

The term membrane encompasses a wide range of potential substrates that can be used for the immobihzation of NAs. It includes traditional polymeric cast membranes , such as nitrocellulose, nylon or polypropylene, and also innovations such as ceramic or track-etched materials (alumina membranes). Membranes as substrates in DNA array fabrication can possess advantages over other surfaces their surface area can be much greater (200% more in cast membranes and 500% more in aliuniniiun membranes) than certain other alternatives. This is primarily a consequence of their possession of pores. 

DNA arrays are fabricated by immobilizing the complementary DNA (cDNA) onto a solid substrate such as silicon, nylon or glass. This can be achieved by robotic printing of polymerase chain reaction (PCR) products (also known as direct-deposition approach), photolithographical synthesis of complementary oligonucleotides or piezoelectric inkjet printing of PCR products (also known as indirect-deposition approach). 

Different types of NA probes can be used in the fabrication of DNA arrays complementary DNA (cDNA), oligonucleotides (OND) and peptide nucleic acids (PNA). 

Classic solid phase substrates used in biotesting, such as microtiter plates, membrane filters or microscope slides, have been the first supports used for NA immobilization in array fabrication [27]. Desired attributes of any DNA array substrate include (i) chemical homogeneity (ii) thermal and chemical stability (iii) ability to control surface chemical properties such as polarity or hydrophobicity (iv) ability to be activated with a wide range of chemical functionalities (v) reproducibihty of the surface modification processes involved (vi) inert with respect to enzymatic activity especially ones involved in DNA manipulation and (vii) ultra-low intrinsic fluorescence. 

Silicon wafers can act as substrates in the fabrication of DNA arrays. Chemical functionalization of silicon surfaces is compUcated by the fact that silicon spontaneously oxidizes in air to produce an amorphous sihca layer. 

All of the supports discussed previously can be used in the fabrication of flat ( positional ) DNA arrays. New approaches to fabricating and applying arrays are continuously being developed, some of which do not rely on... 

DNA arrays have been also generated by Dip-Pen Nanolithography (DPN) [80]. DPN involves the transfer of NAs directly from a coated Atomic Force Microscope (AFM) tip to the substrate of interest by virtue of direct molecular diffusion. Using this technique, thiol-modified ONDs have been patterned onto gold substrates and acrylamide-modified ONDs onto glass sHdes that were previously modified with mercaptopropyltrimethoxysilane. Feature sizes ranging from many micrometers to less than 100 nanometers could be obtained. The deposition of two different OND sequences onto the same substrate has also been reported [80], but the appHcation of this principle to the fabrication of high-density arrays remains to be addressed. 

Cell microarrays have also been fabricated. Ziauddin and Sabatini (2001) demonstrated the ability to transfect cells cultured onto plasmid DNA arrayed in gelatin on a standard DNA microarray slide. Xu (2002) printed down cells in the form of high density microarrays on permeable membranes and demonstrated phenotypic assay performance with the immobilized cells. The commercialization of viable cell arrays will permit an even closer look at cell-mediated events during the drug discovery process. 

Building up expertise in this field, EMBL with its data library is well positioned to play an important European role in estabfishing a reference DNA array and SAGE database. In the future, the micro-array technology will be developed also for proteins, applying miniaturization, nanotechnology, micro-fabricated devices and reaction chambers. 

We further addressed the use of the nucleic acids as biopolymers for the formation of supramolecular structures that enable the electronic or electrochemical detection of DNA. Specifically, we discussed the use of aptamer/low-molecular-weight molecules or aptamer/protein supramolecular complexes for the electrical analysis of the guest substrates in these complexes. Also, nucleic acid-NPs hybrid systems hold a great promise as sensing matrices for the electrical detection of DNA in composite three-dimensional assemblies. While sensitive and selective electrochemical sensors for DNA were fabricated, the integration of these sensor configurations in array formats (DNA chips) for the multiplexed analysis of many DNAs can also be envisaged. 

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Array fabrication Spot morphology, probe solutions (quality and quantity), array design, probing approach Replicate features, reference standards, batch quality control, DNA binding dyes... 

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