DNA microchips

Achieving high fidelity chemical synthesis on glass plates has become increasingly important, since glass plates are substrates widely used for miniaturized chemical and biochemical reaction and analyses. DNA chip can be directly prepared by synthesizing oligonucleotides on glass plates, but the characterization of these micro-syntheses has been limited by the sub-picomolar amount of material available.

MEMS are defined at the micron level by VLSI circuit processing techniques, giving technologists precise control over the finished product. Nanotechnology, which uses the molecular-scale processes of chemistry and living cells, is based on harnessing their molecular interactions to set in motion processes that create some desired end configuration.

One recent result in this direction was a hybrid micromachine/DNA system, announced by IBM researchers at the company's Zurich research center. In the device, tiny micromachined cantilevers were selectively deflected by DNA fragments. The prototype demonstrates for the first time a machine that is driven by DNA molecules. The work, done in tandem with a group at the University of Basil, was reported in a recent issue of the journal Science.

The development of high throughput techniques, such as DNA microarrays, engages interest in many biomedical research fields. They are becoming one of the preferred methods for large-scale expression analyses. The power of this technology is that it allows the profiling of thousands of genes in one single experiment. There are two main array-based technologies: cDNA and oligonucleotide arrays. cDNA arrays consist of microscope slides or nylon membranes containing hundreds to thousands of immobilized DNA probes, which are hybridized to fluorescent or radioactive complementary cDNA obtained from a target sample. Oligonucleotide chips differ in that probes are 20-25 mer selected oligonucleotides, which are bound to glass substrates and that the DNA obtained from a target sample can only be fluorescently labeled. One possible use for the advancements in DNA Microchip fabrication involves strictly computer purposes, and is not thoroughly addressed in this paper. The device uses the lock-and-key mechanism of DNA chemistry. An array of cantilevers is treated with different strands of DNA. When a solution containing different fragments of DNA is introduced, complementary strands of DNA will naturally bind to specific cantilevers. The bonding process creates stress, which deflects the cantilever. The effect has been applied so far to detecting damaged DNA sequences, since a single base mismatch will cause a slightly different stress, indicating the presence of a damaged fragment.

Virtually everything that one wants to know about a cell, including its species, the identity of the individual it came from, whether it is cancerous or normal, whether it should not be where it is (for example a bacterium in a sample of human blood), etc. is contained in the information coded by its DNA. If some of this information could be obtained quickly and easily, there would be almost unlimited opportunities to use it in medical diagnosis, disease treatment, agriculture, forensics, biotechnology, food safety testing, and a host of other areas.

Thanks to recent advances in molecular biology, biochemistry, and microchip manufacturing, extracting this information quickly and inexpensively is not many years away. Already, microchips with 400,000 different DNA spots (probes) are being produced commercially. These can pair up ("hybridize") with DNA or RNA in a liquid sample to learn a great deal about the cell that it came from.

The rapid advance of genome-scale sequencing has driven the development of methods to exploit the information encoded by such genes and to define their participation in physiological and disease processes. Microarray technology seems likely to become a standard tool for both molecular biology research and clinical diagnostics. This could be achieved by the systematic survey of RNA, DNA and even protein variation. The main advantage of this high-throughput method is that it allows generating information of thousands of genes in a single experiment.

DNA microarray is thus the latest in a line of techniques to exploit a potent feature of the DNA duplex: the sequence complimentarity of the two strands. The introduction of solid supports set the trail to array-based methods. The starting point was the observation that single stranded DNA binds strongly to nitrocellulose membranes in a way that prevents the strands from reassociating with each other, but permits hybridization to complementary RNA.

At the present time, the main large-scale application of microarrays is expression analysis. This is followed by the study of DNA variation on a genome-wide scale. Both of these applications have many common requirements, but differ in some important respects that have allowed the development of two different types or arrays. For the analysis of variation, it is important that the reaction forming duplex between target and probe is able to discriminate a single mismatched base pairs, so the high degree of discrimination required is possible only with short probes. Sequence discrimination is less important for the measurement of expression levels, where quantitative measurement over a wide dynamic range is important.

The applications of arrays to genomic studies primarily involve identification and genotyping of mutations. Oligonucleotide microarrays have largely been used for identification of novel DNA variants. With the ability to perform custom synthesis at high density, one can construct a ‘tiling’ array to scan a target sequence for mutations. Each overlapping 25-mer in the sequence is covered by four complementary oligonucliotide probes that differ only by having A, T, C or G substituted at the central position. An amplified product containing the expected sequence will hybridize best to the expect probe, whereas a sequence variation will typically alter the hybridization pattern. Such tiling arrays have been used to detect variants in such targets as the HIV genome, human mitochondria and the gene encoding. In such specific settings, the process can be optimized to have high specificity and sensitivity. The two commonly used types of DNA chips differ in the size of the arrayed nucleic acid components: oligonucleotide chips include short nucleic acids (oligonucleotides up to 25 nucleotides) that can be used for both RNA expression and sequence analysis while cDNA microchips include relatively large nucleic acid components (usually larger than 100 nucleotides) and are often used in RNA expression studies. Basically, oligonucleotide and cDNA arrays consist on glass surfaces, microscope slides or nylon membranes, containing hundreds to thousands of immobilized oligonucleotide or DNA probes respectively. These are hybridized to complimentary labeled cDNA obtained from a target sample, such as cell lines, bacteria, yeast, mouse or human samples. The hybridization signal of complementary cDNAs can be quantified using different detectors depending on the labeling of the target cDNA, namely imaging plate devices for radioactive or laser scanner for fluorescent labeling. The analysis and interpretation of large amounts of data implies the use of appropriate software and bioinformatic tools in conjunction with biological knowledge.

Technology has been moving very rapidly in this field and will in all likelihood continue to do so in the near future. Whatever the kind of chip, four steps are essential for the development of this technology: the fabrication of the microarrays itself, the hybridization process, the reading of the arrays, and the analysis of the data. In the case of oligonucleotide technology, the production of the chips and the instrumentation necessary for their utilization, such as the hybridization chamber, washing and drying stations, scanner and software, are already designed and manufactured by Affymetrix, the main current leader company. The involvement of the user in the design of oligonucleotide chips is increasing. However, the implementation of oligochips implies acquiring simultaneously all the devices specifically designed by the producer necessary for using oligonucleotide chips. On the contrary, different approaches and alternatives can be taken for the development of cDNA microarray technology, depending on the support material were the DNA probes are going to be printed, and the type of labeling to be performed on the target DNA sample. Irrespective of the labeling, it is necessary to use a microarrayer, an optimized hybridization protocol, a radioactivity or fluorescence reader and appropriate software in order to deal with the considerable amount of data generated by the simultaneous analysis of thousands of genes. Microarrayers, scanners or fluorescence or radioactivity detectors, and software can be designed and homemade by the user or purchased to specific companies. The hybridization protocol should be optimized in each laboratory, although different commercial kits facilitate reagents and critical steps in the procedure of making and reading microarrays.

The fabrication of large numbers of DNA microchips has, until recently, been an extremely costly endeavor. Recent breakthroughs in photolithography allow the creation of mask-less chips have greatly reduced costs. Unfortunately, these reductions in manufacturing expenses have not reduced the price of theses chips to the extent that their use may become ubiquitous. Therefore it is imperative that new techniques such as optical lithography be pioneered and explored as possible methods of reducing cost and thereby increasing the availability of DNA microchips. Some of these techniques include GaN light emitting diode displays, as well as the use of florescent lighting to produce Ultra violet light.

The current method of DNA microarray fabrication uses ultraviolet light passed through chrome or glass masks to direct the synthesis of DNA strands composed of photolabile phosphoramidite deoxynucleosides. Photolithographic masks are used to control the light based synthesis. For each nucleotide in length of a DNA strand (N), 4N masks are required to make the chip. A maskless approach would circumvent the need for chrome and glass photolithographic masks, thereby reducing the cost and turnaround time for making custom DNA chips and greatly increasing their versatility.

The need to mass manufacturing of DNA microchips has led to vast amounts of research in optical lithography. Recent breakthroughs have greatly reduced the cost of manufacturing, but future discoveries hold the potential to further reduce manufacturing expenses.

DNA chips are libraries of short strands of DNA designed to identify unknown strands of DNA. When DNA from an outside source is placed on a chip, it seeks to chemically bond with a strand of DNA that is complimentary. Once the two bond, florescent light is emitted through a chemical reaction. Since it is known what the chemical make up of each strand of DNA is on the chip as well as its location, the unknown DNA is easily identified.

To create the libraries on the chips, each strand of DNA must be built nucleotide by nucleotide. In photolithography this feat is accomplished through the use of photo degenerated acids designed to make DNA chemically inert. The photo degenerated acids (PGA) are selectively removed through photo desensitization. DNA is then flushed over the chip and will bond only to the locations that have had the PGA’s removed. Typically Ultra violet light is required to accomplish photo desensitization, and initially, costly masks to direct the light.

Figure 1 diagrams the process of selective photo desensitization. A chip, usually constructed on a glass slide, consists of chemically inert DNA due to bonding with PGA’s. The PGA’s are then selectively removed to allow for the addition of a nucleotide at select sights. A new nucleotide link is added by flushing the chip with that nucleotide. The chip is then flushed with PGA’s again so as to make the entire chip chemically inert once again. The process continues with a new mask each time in order to create a library of DNA strands on a single chip.

A commercial device known as a DNA synthesizer is responsible for the flushing of DNA and PGA’s over the chip. DNA synthesizers are available from several companies; a commonly used example is Expedite 8909; PE Biosystems used by Dr. Xiaolian Gao’s group at the University of Houston. In order to create all possible strands of DNA for a given length (x) using masks, a technique known as binary masking is used. Binary masking is the creation of masks to selectively photo desensitize, in order to systematically create an established library of DNA strands. If a chip is to have all combinations of DNA that are 10 nucleotides long it would require 2^x or 2^10 different strands of DNA. To create the chip 40 costly masks would be required. This was a great drawback to DNA microchip manufacturing that required masks.

Furthermore, such masks would be vulnerable to damage. An imperfection comparable to a basketball in a state the size of Maryland will result in an inoperable DNA chip. Another concern is the increasing difficulty induced through attempting to increase resolution. As a result mass production using masks has a dim future.

As time progressed it was discovered that the need for masks could be eliminated, through the use of micro mirror arrays controlled by computers. Texas Instruments pioneered the micro mirror array and had great success. They were able to produce DNA chips at much lower cost, because they had eliminated the need for masks. Their progress is a milestone in photolithography. Unfortunately the micro mirror apparatus is relatively costly, and therefore was unable to reduce the cost of DNA chips so that their use could become commonplace.

Further disadvantages stem from the comparatively low resolution accompanying micro mirror arrays. Texas Instruments Digital Micromirror array consists of an array of mirrors 600 x 800 16 micrometers square independently controlled mirrors. As a result, desensitization can only occur in areas that are 16 x 16 micrometers, many molecules in diameter, making the practicality of micromirror arrays rather questionable. Texas Instruments recognized this fact and has moved away from micro mirror array selective photodesensitization in favor of using the technology for high definition projection devices in which 16-micrometer pixelation is an exceptional accomplishment. Further work may eventually bleed over into the overall television industry, but it is more likely that basic nanotubules will prove more efficient for high definition television systems.

However, the use of micro mirror arrays has reduced the cost of certain DNA chips making the technology a success, despite the disadvantage of poor resolution. Recent declines in DNA microchip price are reflected in the use of Texas Instruments micromirror apparatus.

Another recent discovery involves the use of lithographically induced self-construction of polymer microstructures for resistless patterning. Methods have been developed that can directly pattern polymer microstructures of arbitrary shapes without using a resist, exposure, chemical development, and etching. A mask with protruded atterns is placed above an initially flat polymer film cast on a substrate. When the polymer is heated above its glass transition temperature, the polymer would assume the structure of the pattern when cooled back to room temperature.

The advantages to this technique include the minimizing of tedious steps required in traditional techniques, as well as a significant reduction in the number of masks necessary to make oligonucleotides that are a particular number of mers long. Unfortunately the technique cannot adequately be applied to DNA microchip synthesis, because the process is only capable of causing a polymer to assume a particular shape, not selectively desensitize a chip for the next level of coupling. As a result of the nature of the process, resistless lithographic techniques is not a viable alternative to traditional lithographic techniques. Researchers have begun to pioneer new methods of optical photodesensitization that would be less costly than both masks and micro mirror arrays. Some possibilities include the use of LED’s in the Ultra violetA spectrum as well as florescent lighting in order to induce photo desensitization.

Presently there are very few semiconductors with the necessary band gap and wavelength in order to desensitize the DNA strands. Band gap is directly correlated with the number of electron volts emitted when a current is passed through the semiconductor. Electron volts (EV) are the amount of photonic energy emitted from the semiconductor. Larger band gaps cause larger quantities of energy to be released.

One possibility is the newly developed Gallium Nitride (GaN). GaN is currently being grown using Metal Organics Vapor Phase Epotaxy (MOVPE) and has proven itself as a powerful Ultra violet LED. MOVPE is the process by which crystals, often semiconductors, are artificially produced by controlling impurities in crystalline structures at the microscopic level. The process is considerably new and has already produced a wide range of new semiconductors, many with unique properties. GaN is the one of the only semiconductors that emits light in the near Ultra violet and blue light spectrum with over 3eV of energy. Currently, LED displays are used in computers and televisions using semiconductors that emit light in the visible spectrum. However, with the recent creation of GaN, it is possible that similar techniques will be used in order to photo desensitize DNA chips. This avenue holds great potential as an extremely inexpensive method to manufacture DNA chips. Another possibility includes the use of Ultra violet sources similar to florescent lighting. A typical florescent light is composed off a glass tube low-pressure mercury vapor inside. The electrons in the vapor are excited via electrical current and will emit light in the Ultra violet spectrum. However, the glass tube is coated with a phosphor that absorbs the Ultra violet light and emits in the visible spectrum.

A device similar to a florescent light called a germicidal lamp produces light in the Ultra violet spectrum. A germicidal lamp, often called a mercury lamp, is constructed using thinner glass and does not have a phosphor coating. As a result, deep Ultra violet light is emitted from this type of lamp. Another option is to continue to use a phosphor, but one that emits in the near Ultra violet spectrum as opposed to the visible light spectrum.

Such lamps are commonly referred to as black-light lamps. Black light falls between the visible spectrum and the Ultra violet spectrum on the electromagnetic spectrum. Typical wavelengths are between 300 and 400 nm. Slightly below 400 nm is ideal for selective photo desensitization, and therefore holds great potential for use in the mass fabrication of DNA microchips.

It is inevitable that new methods of producing DNA chips must be pioneered. Although micro mirror arrays have reduced the cost of DNA chips, it has not been enough to make such microchips available to the general public. Furthermore, if microchip production methods are simplified and become considerably less expensive, it is possible for many scientists to produce their own DNA chips.

If the uses of LED displays are incorporated into the manufacturing process, it is possible that microchip costs will decrease. It may also be possible that the difficulty of manufacturing be alleviated, thereby increasing the number of businesses and people capable of producing said chips.

Living cells, while employing chemical reactions, are in some ways closer to mechanochemistry in that really large molecules like DNA, RNA or proteins drive the reaction. However, as Ehr pointed out, these reactions take place close to thermodynamic equilibrium, which limits their range of action. Mechanochemistry operates far from equilibrium, producing fast, high-energy reactions that could forge fundamentally new types of molecular machines.

Such micron-scale machine tools, using probe techniques borrowed from microscopy, may become the enabling technology for building nanoscale systems. For example, the machines could be employed to build specific complex molecules that could be the basic building blocks of what Ehr defines as mechanochemistry. This would be a new variant of chemical manufacturing in which specifically tailored molecules are positioned within a reaction and mechanical or electrical force is applied to drive constituents of the reaction together. Conventional chemistry relies on the diffusion of molecules through the reaction, as the principal mechanical component that drives chemical synthesis.

A prototype nanomanipulator has been built to perform operations in the vacuum chamber of an electron microscope. Using tiny micromachine actuators driving atomic-force microscope (AFM) tips, the tool can build objects out of carbon nanotubes. One of the first tasks of the manipulator will be building better AFM tips. A group at Washington University (St. Louis, Mo.) is studying the possible operations that can be performed with the machine.

A new development in the photolithography method has been the use of computer-controlled micromirror arrays to direct the light to the desired positions on the chip, instead of using masks. This new method could prove to be of very high commercial value, as production costs are greatly reduced, since new masks are not required for every new set of microarray probes.

Another in-situ synthesis method is the ink-jet style in-situ synthesis method developed by Rosetta and Agilent. In this method, standard dimethoxytrityl blocked phosphoramidites are used to construct oligonucleotides. Compared to the Affymetrix photolithography method, the step wise coupling efficiency is higher and therefore the quality of the oligonucleotides produced on the chips is better. Also, this methodology is much more flexible than the Affymetrix method, so that it is more useful for the researcher who needs to frequently change the design of the oligonucleotides on the chips.

Another advantage is that reverse amidites (3'-dimethoxytrityl-blocked 5'-phosphoramidites rather than 5'-dimethoxytrityl-blocked 3'-phosphoramidites) can be used to make oligonucleotides with free 3'-OH groups. Therefore, this kind of chip can be used for direct hybridisation assays or for assays using extension of the primer by polymerase enzymes after hybridisation to the target DNA (Arrayed Primer Extension (APEX) assays).

In standard DNA synthesis, the phosphoramidite molecule is situated at the 3' position of the ribose ring. The dimethoxytrityl (DMT) group is therefore located at the 5' position of the ribose ring. Thus synthesis takes place in a 3' to 5' direction, unlike enzymatic synthesis which proceeds in a 5' to 3' direction. As already noted, the use of reverse phosphoramidites allows synthesis to proceed in a 5' to 3' direction as the cyanoethyl phosphoramidite group is located at the 5' position and the dimethoxytrityl is located at the 3' position. Oligonucleotides generated using standard DNA synthesis instrumentation are formed with a hydroxyl moiety at the 3' and 5' termini. Any additional groups, such as phosphates, biotin, amino linkers, or thiol linkers, must be added as a separate modification step. Thus, these commonly requested 'modifications' (that can be used to produce microarrays) are also available as phosphoramidites for use directly on the automated synthesizer.

In conclusion, researchers should begin to research photo desensitization through Ultra violet LED’s. Through MOVPE new, and more reliable semiconductors with larger band gaps are being developed. These new semiconductors must be tested and their usefulness ascertained, so that new methods of selective photo desensitization may be pursued. As more efficient means of photo desensitization are established, DNA microchips will lower and cost, and become a more available resource to doctors and hospitals.