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New Electrostatic-based DNA Microarray Technique

The dream of personalized medicine — in which diagnostics, risk predictions and treatment decisions are based on a patient's genetic profile — may be on the verge of being expanded beyond the wealthiest of nations with state-of-the-art clinics. A team of researchers with the U.S. Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab) has invented a technique in which DNA or RNA assays — the key to genetic profiling and disease detection — can be read and evaluated without the need of elaborate chemical labeling or sophisticated instrumentation. Based on electrostatic repulsion — in which objects with the same electrical charge repel one another — the technique is relatively simple and inexpensive to implement, and can be carried out in a matter of minutes.

"One of the most amazing things about our electrostatic detection method is that it requires nothing more than the naked eye to read out results that currently require chemical labeling and confocal laser scanners," said Jay Groves, a chemist with joint appointments at Berkeley Lab's Physical Biosciences Division and the Chemistry Department of the University of California (UC) at Berkeley, who led this research. "We believe this technique could revolutionize the use of DNA microarrays for both research and diagnostics."

Groves, who is also a Howard Hughes Medical Institute (HHMI) investigator, and members of his research group Nathan Clack and Khalid Salaita, have published a paper on their technique in the journal Nature Biotechnology, which is now available online. The paper is entitled "Electrostatic readout of DNA microarrays with charged microspheres."

In their paper, Groves, Clack, and Salaita describe how dispersing a fluid containing thousands of electrically-charged microscopic beads or spheres made of silica (glass) across the surface of a DNA microarray and then observing the Brownian motion of the spheres provides measurements of the electrical charges of the DNA molecules. These measurements can in turn be used to interrogate millions of DNA sequences at a time. What's more, these measurements can be observed and recorded with a simple hand-held imaging device — even a cell phone camera will do.

"The assumption has been that no detection technique could be more sensitive than fluorescent labeling, but this is completely untrue, as our results have plainly demonstrated," said Groves. "We've shown that changes in surface charge density as a result of specific DNA hybridization can be detected and quantified with 50-picomolar sensitivity, single base-pair mismatch selectivity, and in the presence of complex backgrounds. Furthermore, our electrostatic detection technique should render DNA and RNA microarrays sufficiently cost effective for broad world-health applications, as well as research."

Your susceptibility to a given disease and how your body will respond to drugs or other interventions is unique to your genetic makeup. Under a personalized medicine plan, treatment effectiveness is maximized and risks are minimized by tailoring disease treatments specifically to you. This requires the precise diagnostic tests and targeted therapies that can stem from assays using a DNA microarray — a thumbnail-sized substrate containing thousands of microscopic spots of oligonucleotides (stretches of DNA about 20 base pairs in length) laid out in a grid.

Often referred to as "gene chips," DNA microarray assays and their RNA counterparts have become one of the most powerful tools for gene-expression profiling, the identification of mutations, and the detection of multiple pathogens in patients afflicted either by multiple diseases or drug-resistant strains of diseases. Aside from their potential future role in personalized medicine, the widespread use of DNA microarray assay devices could have an immediate and profound impact on the treatment of diseases today. For example, according to a report two years ago from the Global Health Diagnostics Forum, 400,000 lives could be saved each year from death by tuberculosis through the use of DNA microarray assays rather than the standard TB diagnostic test.

We have demonstrated parallel sampling of a microarray surface with micron-scale resolutions over centimeter-scale lengths," said Groves. "This is four orders of magnitude larger than what has been achieved to date with conventional scanning-electrostatic-force microscopy."

In a typical experiment, a microarray is prepared and mounted in a well chamber and the DNA is hybridized (a standard technique in which complementary single strands of DNA bind to form double-stranded DNA "hybrids"). A suspension of negatively-charged silica microspheres is then dispersed through gravitational sedimentation over the microarray surface, a process which takes about 20 minutes. Because the substrate or background surface of the microassay is positively charged, the silica microspheres will spread across the entire surface and adhere to it. However, on surface areas containing double-stranded DNA, which is highly negatively charged, and on areas containing single-stranded DNA, also negatively charged but to a lesser degree than double-stranded DNA, the microspheres will levitate above the substrate surface, stacking up in "equilibrium heights" that are dictated by a balance between gravitational and electrostatic forces.

These electrostatic interactions on the microarray surface result in charge-density contrasts that are readily observed. Surface areas containing DNA segments take on a frosted or translucent appearance, and can be correlated to specific hybridizations that reveal the presence of genes, mutations and pathogens.

There are a number of short-term "next steps" for this research, Groves said, including testing its application in high-density arrays and pushing its ultimate resolution limits.

"Since the resolution of electrostatic-based imaging is determined by the number of particle-observations rather than by the diffraction limit of light, our readouts could serve as a form of ultramicroscopy," he said. "The real grand challenge for this technology, however, will be for us to find suitable industrial partners with whom we can work to see that useful new products actually make it to market.

DNA Microarray picture

A turning point in Science and our approach to the human body occurred in 1953 when Watson and Crick discovered the double helix structure of DNA. DNA consists of four base chemicals called nucleotides: adenine, guanine, cytosine and thymine that reside on the inner surface of two parallel sugar and phosphate rails. DNA holds all the information required to produce and maintain a living organism.

In 2003 the Human Genome Project was completed and the entire sequence of human genes was unraveled, mapped and stored in databases. In the past decade DNA microarray technology has made it possible to measure the expression levels of many thousands of genes simultaneously.

DNA microarrays are quite complex, but basically thousands of microscopic spots of DNA can be attached to a solid surface, usually glass or silicon. This gives rise to the name “gene chip”. A gene chip can be analyzed for coding structure. The coding structure can then be referenced against the knowledge that exists on the gene databases. The genes can also be referenced to a person’s own DNA to observe changes and differences over time.

A recent article in the Australian Journal of Clinical Hypnotherapy & Hypnosis made me aware of the existence of a branch of research entitled Psychosocial Genomics. Psychosocial Genomics is the study of how inner psychological events and interactive social events can stimulate gene expression. The process is thought to proceed from brain to body.

Until I read this article I had no idea that such research has ever taken place and I was immediately fascinated by it. I had been practicing Clinical Hypnotherapy since 1993, knowing that it achieved amazing results but not thoroughly understanding how these results were achieved. I had too much scientific training behind me to be caught up in the egoistic notion that it was just I who was responsible for the results. I knew instinctively that changes must take place in the clients’ neurotransmitters levels, but further than that, did not give it further thought.

Reading the article revealed to me that Ernest Rossi, a leading Psychotherapist and Clinical Psychologist had proposed a connection between gene expression, protein synthesis and hypnosis as far back as the mid-seventies.

Psychosocial Genomics, through the examination of gene expression seeks to understand the activity of the hypnotherapeutic process and provide some degree of measure. Gene expression enables an ongoing cascade of processes in the body including protein synthesis, and a swathe of biological processes. The biological processes include positive healing activities of the immune system (anti-inflammatory), beneficial alteration of mental and emotional states (hormonal, peptide and neurotransmitter balance) and the growth and development of new, neural and neuronal structures (nerve and brain plasticity.

DNA Microarrays Methods Express, Mark Schena, Ph.D.

DNA Microarrays – Methods Express, edited by Mark Schena and published by Scion Publishing. Methods book containing 12 chapters from leading laboratories covering the latest protocols and procedures for DNA microarray experimentation. This must-have book is an essential resource for all laboratories working on DNA microarrays.

Electronic DNA microarrays

DNA contains and issues the language of life. It gives cells instructions for living, and tells living organisms about their hereditary traits. This language is coded into the DNA’s famous double helix structure: Fig 1(a). Each helical strand exhibits a sequence of four chemical bases, adenine (A), guanine (G), cytosine (C), & thymine (T), e.g., CAAGTG. The two twisted strands are bound together by pairing base A always with base T, and G always with C. Due to this pairing rule, within any section of DNA, once the sequence of one strand is identified, that of the other strand is easily inferred: e.g., the complementary sequence of CAAGTG is GTTCAC. DNA sequences are the language of life. Reading them, therefore, is of prime importance.
    Electronic DNA microarrays are CMOS integrated circuits (ICs) that can rapidly decipher unknown DNA sequences [1-6]. A double-stranded DNA molecule can unzip into two complementary strands. A single-stranded DNA molecule thus obtained can bind back to its complementary sequence (either the old mate or a new one), forming again a double-stranded DNA molecule. This binding of two complementary strands, or hybridization, underlies the genetic sequencing operation of the electronic DNA chip.

The electronic DNA microarray is constructed by immobilizing single-stranded DNA molecules of different identified sequences onto different grid points on a CMOS IC [Fig. 1(b)]. The grid points are often defined by post-fabricated gold electrodes that are electrically connected to the underlying CMOS IC. Different grid points represent distinct DNA sequences. These single-stranded DNA molecules of known sequences making up the array are called DNA probes. Now consider single-stranded DNA molecules of an unknown sequence, or DNA targets. When a solution of DNA targets is introduced onto the DNA array, the target strands wander around to eventually hybridize to their complementary probe strands at a specific grid point [Fig. 1(b)]. Locating the hybridization position reveals the target sequence, for we already know the probe sequence at that position, which must be complementary to the target sequence. The CMOS IC underneath is used for electronic detection of the hybridization point.
One well-established electronic detection technique is to label DNA targets with reporter molecules of a distinctive electronic signature and to search for them. Redox enzymes are an example of such electronic labels. If a voltage is suddenly applied between an electrode where hybridization occurred and the electrolyte (DNA target solution), redox labels attached to target molecules will give up electrons to the electrode, thereby increasing the current through the electrode. The same voltage step in an electrode with no hybridization would cause no such current increase as redox labels are absent at that electrode. By applying the voltage step and monitoring current change fast across the whole array using the underlying IC, hybridization positions are rapidly detected, leading to target sequence identification. Redox-label-based CMOS DNA chip examples are found in [1-3].
While the label-based detection offers excellent sensitivity, significant efforts are being placed to develop label-free electronic DNA microarrays [4,5] because elimination of labeling steps would simplify the sample preparation. In [4], for example, the capacitance of an electrode immersed in the electrolyte is monitored to sense hybridization. No labeling is needed, as target strands added to an electrode during hybridization naturally lead to a dielectric constant change, or, capacitance change.
Field effect transistors (FETs), the basic commodity of CMOS ICs, can be also useddirectly for label-free electronic detection of DNA hybridization [7-9]. Underlying this sensing modality is the exploitation of the impact of DNA’s intrinsic negative charges upon the FET behavior. Imagine that underneath the DNA array there is a corresponding FET array integrated in the CMOS IC. The gate dielectric of each FET is linked to DNA probe strands of the same sequence in a corresponding site in the DNA array. When a target strand hybridizes to its complementary probe strand anchored to a specific FET gate, the target’s intrinsic charge alters the channel conductance and capacitance of the FET. Therefore, by monitoring the channel of each FET in the array, one can attain label-free electronic readout of target sequences. While field effect sensorsare widely used [9], there is a lot of room for development in their use in DNA microarrays. Post-processing to expose gate dielectrics to electrolyte may pose a challenge. The smallest possible FET width must be used to maximize the impact of the DNA’s charge on channel properties.
Hybridization is at the heart of many other DNA sequencing techniques. What makes electronic DNA microarrays unique is their massively parallel operation. Distinct probe sequences numbering as many as hundreds of thousands can be simultaneously available across an array. The CMOS IC monitors each site of the array fast across at a gigahertz speed, and hence, its operation may be regarded as parallel to human eyes. This parallelism allows for rapid collection of vast amounts of genetic information (far faster than non-microarray techniques), accelerating the speed at which we probe the secrets of living organisms. The parallelism is a direct outcome of using CMOS microfabrication techniques to build large microarrays, and is enhanced by the use of integrated electronics.
In the original invention, which is still the commercially dominant form of the DNA microarray, hybridization is sensed by optical means [10]: florescent dyes labeling target strands light up upon illumination, reporting hybridization points. This optical machine boasts sensitivity superior to, and parallelism similar to, its much smaller electronic cousin. Although considerable work is needed to develop a high-performance electronic DNA chip as an alternative to the optical type, decisive advantages of using ICs (small size, low cost, programmability, real-time, label-free options) are the cogent reason for the ever-growing efforts in the development of electronic DNA chips.


Other electronic biosensor microarrays

Generalizing the concepts of the electronic DNA chip, one can readily consider CMOS biosensors that can detect other biological objects such as viruses and disease marker proteins [11]. Just like a DNA strand sticks specifically to its complementary strand, a virus or a protein binds specifically to its unique biochemical mate, an antibody. This highly specific binding is analogous to the way different keys fit into different locks. Fig. 2 illustrates a scenario where nine different types of viruses can be detected by using an array of nine specifically corresponding types of antibodies. When the array is exposed to a target solution containing type-4 viruses, for instance, the viruses will specifically bind to antibodies in array point 4. This binding can be detected using the CMOS IC below in much the same ways DNA hybridization is detected. By reading the location of the binding, the presence and type of viruses are determined. The multiplexed array platform can be especially useful for the diagnostics of complex diseases like cancer.

Electronic DNA microarrays

DNA contains and issues the language of life. It gives cells instructions for living, and tells living organisms about their hereditary traits. This language is coded into the DNA’s famous double helix structure: Fig 1(a). Each helical strand exhibits a sequence of four chemical bases, adenine (A), guanine (G), cytosine (C), & thymine (T), e.g., CAAGTG. The two twisted strands are bound together by pairing base A always with base T, and G always with C. Due to this pairing rule, within any section of DNA, once the sequence of one strand is identified, that of the other strand is easily inferred: e.g., the complementary sequence of CAAGTG is GTTCAC. DNA sequences are the language of life. Reading them, therefore, is of prime importance.
    Electronic DNA microarrays are CMOS integrated circuits (ICs) that can rapidly decipher unknown DNA sequences [1-6]. A double-stranded DNA molecule can unzip into two complementary strands. A single-stranded DNA molecule thus obtained can bind back to its complementary sequence (either the old mate or a new one), forming again a double-stranded DNA molecule. This binding of two complementary strands, or hybridization, underlies the genetic sequencing operation of the electronic DNA chip.

The electronic DNA microarray is constructed by immobilizing single-stranded DNA molecules of different identified sequences onto different grid points on a CMOS IC [Fig. 1(b)]. The grid points are often defined by post-fabricated gold electrodes that are electrically connected to the underlying CMOS IC. Different grid points represent distinct DNA sequences. These single-stranded DNA molecules of known sequences making up the array are called DNA probes. Now consider single-stranded DNA molecules of an unknown sequence, or DNA targets. When a solution of DNA targets is introduced onto the DNA array, the target strands wander around to eventually hybridize to their complementary probe strands at a specific grid point [Fig. 1(b)]. Locating the hybridization position reveals the target sequence, for we already know the probe sequence at that position, which must be complementary to the target sequence. The CMOS IC underneath is used for electronic detection of the hybridization point.
One well-established electronic detection technique is to label DNA targets with reporter molecules of a distinctive electronic signature and to search for them. Redox enzymes are an example of such electronic labels. If a voltage is suddenly applied between an electrode where hybridization occurred and the electrolyte (DNA target solution), redox labels attached to target molecules will give up electrons to the electrode, thereby increasing the current through the electrode. The same voltage step in an electrode with no hybridization would cause no such current increase as redox labels are absent at that electrode. By applying the voltage step and monitoring current change fast across the whole array using the underlying IC, hybridization positions are rapidly detected, leading to target sequence identification. Redox-label-based CMOS DNA chip examples are found in [1-3].
While the label-based detection offers excellent sensitivity, significant efforts are being placed to develop label-free electronic DNA microarrays [4,5] because elimination of labeling steps would simplify the sample preparation. In [4], for example, the capacitance of an electrode immersed in the electrolyte is monitored to sense hybridization. No labeling is needed, as target strands added to an electrode during hybridization naturally lead to a dielectric constant change, or, capacitance change.
Field effect transistors (FETs), the basic commodity of CMOS ICs, can be also useddirectly for label-free electronic detection of DNA hybridization [7-9]. Underlying this sensing modality is the exploitation of the impact of DNA’s intrinsic negative charges upon the FET behavior. Imagine that underneath the DNA array there is a corresponding FET array integrated in the CMOS IC. The gate dielectric of each FET is linked to DNA probe strands of the same sequence in a corresponding site in the DNA array. When a target strand hybridizes to its complementary probe strand anchored to a specific FET gate, the target’s intrinsic charge alters the channel conductance and capacitance of the FET. Therefore, by monitoring the channel of each FET in the array, one can attain label-free electronic readout of target sequences. While field effect sensorsare widely used [9], there is a lot of room for development in their use in DNA microarrays. Post-processing to expose gate dielectrics to electrolyte may pose a challenge. The smallest possible FET width must be used to maximize the impact of the DNA’s charge on channel properties.
Hybridization is at the heart of many other DNA sequencing techniques. What makes electronic DNA microarrays unique is their massively parallel operation. Distinct probe sequences numbering as many as hundreds of thousands can be simultaneously available across an array. The CMOS IC monitors each site of the array fast across at a gigahertz speed, and hence, its operation may be regarded as parallel to human eyes. This parallelism allows for rapid collection of vast amounts of genetic information (far faster than non-microarray techniques), accelerating the speed at which we probe the secrets of living organisms. The parallelism is a direct outcome of using CMOS microfabrication techniques to build large microarrays, and is enhanced by the use of integrated electronics.
In the original invention, which is still the commercially dominant form of the DNA microarray, hybridization is sensed by optical means [10]: florescent dyes labeling target strands light up upon illumination, reporting hybridization points. This optical machine boasts sensitivity superior to, and parallelism similar to, its much smaller electronic cousin. Although considerable work is needed to develop a high-performance electronic DNA chip as an alternative to the optical type, decisive advantages of using ICs (small size, low cost, programmability, real-time, label-free options) are the cogent reason for the ever-growing efforts in the development of electronic DNA chips.


Other electronic biosensor microarrays

Generalizing the concepts of the electronic DNA chip, one can readily consider CMOS biosensors that can detect other biological objects such as viruses and disease marker proteins [11]. Just like a DNA strand sticks specifically to its complementary strand, a virus or a protein binds specifically to its unique biochemical mate, an antibody. This highly specific binding is analogous to the way different keys fit into different locks. Fig. 2 illustrates a scenario where nine different types of viruses can be detected by using an array of nine specifically corresponding types of antibodies. When the array is exposed to a target solution containing type-4 viruses, for instance, the viruses will specifically bind to antibodies in array point 4. This binding can be detected using the CMOS IC below in much the same ways DNA hybridization is detected. By reading the location of the binding, the presence and type of viruses are determined. The multiplexed array platform can be especially useful for the diagnostics of complex diseases like cancer.

A new threat

Imagine this: at the Olympic games in London in 2012, a little known athlete surprises everyone in the men’s 100 metres, thrashing the established favourite by almost a whole second. “Is he on drugs?” everyone asks. Tests find nothing, and he is labelled a freak of nature. But are his achievements thanks to natural ability or are they down to genetic modification?

In our darkest memories, we can all recall having lied, cheated, or stretched the rules, often to gain a personal advantage. For elite athletes the pressures that drive cheating are vastly increased. Sport requires that participants train hard over long periods, often from a young age, linking achievement to self esteem, emotional state, and lifestyle. The temptations of money and fame and pressure from parents, coaches, and fans drive athletes to accept extreme risk to gain even small advantages.

Perhaps it is unsurprising that some athletes turn to science and drugs to improve their chances. In 1967 the British cyclist Tom Simpson collapsed and died on the slopes of Mont Ventoux, in France, largely because of misuse of amphetamines. As tests were developed a number of high profile cases brought doping into the public eye: Ben Johnson was stripped of his gold medal in the 1988 Olympic games, and the whole Festina team was ejected from the Tour de France in 1998 after doping equipment was found in the car of a team official.

An independent international body was established to fight doping on a global scale—the World Anti-Doping Agency. It updates the world antidoping code, which contains a list of prohibited substances and practices; coordinates the testing and education of athletes and their coaches; and funds research into testing to remain one step ahead of potential cheats.

In 2002 the agency recognised a new threat—“gene doping”—by including it in the list of prohibited practices, defined as “the non-therapeutic use of cells, genes, genetic elements, or of the modulation of gene expression, having the capacity to enhance athletic performance.

The techniques involved in gene doping are spawned from the techniques of gene therapy, in particular gene transfer with viral vectors (fig 1). The aim of gene manipulation in these two settings can be clearly distinguished. In gene therapy the goal is treatment and requires the replacement of a defective gene or expression of a therapeutic gene. Gene doping, however, aims to improve athletic performance by increasing or decreasing production of endogenous molecules.

Microarray approaches to finding protein–DNA binding

Mapping protein localization to DNA using chromatin immunoprecipitation and DNA microarrays^32, 33. In this technique, proteins are crosslinked to genomic DNA in a cellular context. The DNA is sheared or digested, leaving DNA–protein complexes that can be precipitated using protein-specific antibodies. Two pools of DNA fragment — those released from the protein and those from control DNA — are amplified and labelled using different fluorescent dyes. Both sets of probes are simultaneously hybridized to a DNA microarray that contains intragenic DNA sequences. The greater the difference in the fluorescent intensity at any fragment on the array, the stronger the binding of the protein to that fragment. b | Mapping protein localization to DNA using DNA adenine methyltransferase identification³⁴. In this method, the DNA adenine methyltransferase enzyme is fused to a chromatin-associated protein and is expressed in cells; the chimeric protein binds to chromatin and methylates adenine residues in the vicinity of the protein-binding site. The methylation-specific restriction enzyme Dpn1 recognizes and cleaves the DNA at methylated GATC sites. The resulting fragments are fractionated by size, then labelled and probed to the array.

Technologies MicroArray

2 samples of RNA are taken from 2 seperate sources and cDNA is created from each by using a technique called RT-PCR (Reverse Transcriptase Polymerase Chain Reaction). Each cDNA sample is labeled with its own specific dye. Both cDNA samples are then washed over a single array, after which dye intensities on each spot is calculated using a fluorescence camera.
Aging
-The free radical theory of aging
-Mitochondria and aging
-The glycation theory of aging
-Proteins damage and maintenance in aging
-Dna damage and dna repair
-Telomeres and aging
-Cellular senescence and apoptosis in aging
-Longevity genes
-Gene silencing in aging
-Hormones and aging
-The immune system and aging
-Inflammation and aging
-Accumulation of toxins and chemical garbage
-Cancer and aging
-Biomarkers of aging
-Caloric restriction with adequate nutrition

Detailed cDNA Microarray Technology Scheme

DNA microarrays work on the principal of base-pairing (See: Basic Biology, base-pairing). Base-pairing allows probes to hybridize to targets on the microarray. (See Basic Biology, hybridization).

At a basic level microarrays are implemented as follows: a cell's RNA is extracted. This RNA (targets) is then multiplied, labeled with fluorescence and hybridized to existing DNA (probes) on the microarray. After hybridization, the probes that were hybridized with targets are fluorescent and a computer scanner is able to detect this fluorescence. Those probes that are fluorescent correspond to the genes that were expressed in the cell.

The microarray is composed of millions of spots (sometimes referred to as cells), each with thousands of probes. Each 20 micrometer cell can contain up to 10^7 probes. The enormous number of probes is to increase hybridization probability and possibilities. Figure 1 is an illustration of the principal of hybridization. A cell (G) is laced with probes of DNA (either oligonucleotide sequences, or cDNA. See below for more information). The fluorescent targets (green circles) are then exposed to the microarray and allowed to hybridize.

Experiments using cDNA microarrays typically involve two cells: a control cell and an experimental cell. First, using robotics, the microarray is laced with DNA probes corresponding to the genes of interest (or the entire organism's genome).

Second, mRNA from a control cell and an experimental cell is isolated. This mRNA corresponds to the genes in the cell that are being expressed (See basic biology, Why study genes). Using reverse transcriptase , the mRNA is then converted to cDNA . The cDNA from both cells are labeled, using fluorescence, different colors. This fluorescent dye can be identified by a computer scanner. The labeled cDNA is considered the target, which hybridizes via base-pair interactions with the probe. (See Basic Biology: Hybridization)

Once the targets are exposed to the microarray for a sufficient amount of time to allow for hybridization (typically 12-16 hours), the array is washed. Certain probes on the array will now be fluorescent, because the fluorescent targets have hybridized to them.

Only the probes containing genes that have been transcribed in the cell will be fluorescent. Those genes that are being expressed in the control cell will fluoresce one color (green), while those expressed in the experimental cell will fluoresce another color (red). Those genes expressed in both cells will have a mixed color (yellow). The amount each gene is being expressed can also be measured by how intense the fluorescence is.

A computer is used to measure the intensity and color of each and every spot on the microarray. Software can then be used to produce data on exactly which genes are being expressed in the cells, and how much each of those genes is being expressed.

Oligonucleotide Arrays

Oligonucleotide arrays are commonly used in biology laboratories and in clinical research projects. The Affymetrix GeneChip is the most widely used oligonucleotide array. Whereas the aforementioned cDNA microarrays use long strands of DNA as fixed probes, the GeneChip uses oligonucleotide sequences as its probe. The whole genome of an organism can be placed on a single microarray as oligonucleotide probes. These oligonucleotide sequences are usually around 25 base pairs in length (see Basic Biology: Base Pairs) (Ref. #7). Below is a explanation of how Affymetrix's technology works.

In order to use the array, first mRNA is extracted from a cell and reverse transcriptase is applied to obtain cDNA. In oligonucleotide arrays, in vitro transcription (for more information on in vitro transcription, click here) occurs to obtain biotin labeled cRNA (See Basic Biology: Base Pairing with RNA). These cRNA molecules are then exposed to the microarray. Overnight, the cRNA molecules (the targets) hybridize to the oligonucleotide probes. After hybridization, the chip is stained with a fluorescent molecule (streptavidin-phycoerythrin) that binds to biotin. The staining protocol includes a signal amplification step that employs anti-Streptavidin antibody and biotinylated goat IgG antibody (The series of washes and stains with aforementioned reagents binds the biotin and provides an amplified flour that emits light when the chip is then scanned with a confocal laser and the distribution pattern of signal in the array is recorded (Ref 53)) A scanner analyzes the GeneChip for signals. Advanced algorithms are then used to give data on the expression levels of the genes of interest.

For a detailed explanation of how the entire process of using Affymetrix GeneChips, including RNA extraction and amplification, is currently being implemented in laboratories see Gene Expression Studies.

Other Variations

One great advantage of microarrays is their flexibility. Many different platforms exist, and many more can be created. The two platforms outlined above may be modified as needed by a researcher. For example if a particular experiment requires the use of DNA as a target instead of RNA it could be easily implemented.

For clinical use, the most important microarray to date is the Roche AmpliChip CYP450. The AmpliChip CYP450 is “world's first pharmacogenomic (See Pharmacogenomics) microarray designed for clinical applications.” (Ref. 52)The chip is based on Affymetrix GeneChip technology, but is designed specifically for clinical use.

Other commercial variations are also found and include Nanogen's NanoChip.

DNA microarray

A DNA microarray is a multiplex technology used in molecular biology. It consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features, each containing picomoles (10−12 moles) of a specific DNA sequence, known as probes (or reporters). These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target. Since an array can contain tens of thousands of probes, a microarray experiment can accomplish many genetic tests in parallel. Therefore arrays have dramatically accelerated many types of investigation

Techniques of molecular biology

Since the late 1950s and early 1960s, molecular biologists have learned to characterize, isolate, and manipulate the molecular components of cells and organisms. These components include DNA, the repository of genetic information; RNA, a close relative of DNA whose functions range from serving as a temporary working copy of DNA to actual structural and enzymatic functions as well as a functional and structural part of the translational apparatus; and proteins, the major structural and enzymatic type of molecule in cells.

Macromolecule blotting and probing

The terms northern, western and eastern blotting are derived from what initially was a molecular biology joke that played on the term Southern blotting, after the technique described by Edwin Southern for the hybridisation of blotted DNA. Patricia Thomas, developer of the RNA blot which then became known as the northern blot actually didn't use the term. Further combinations of these techniques produced such terms as southwesterns (protein-DNA hybridizations), northwesterns (to detect protein-RNA interactions) and farwesterns (protein-protein interactions), all of which are presently found in the literature.

Polymerase chain reaction

The polymerase chain reaction is an extremely versatile technique for copying DNA. In brief, PCR allows a single DNA sequence to be copied (millions of times), or altered in predetermined ways. For example, PCR can be used to introduce restriction enzyme sites, or to mutate (change) particular bases of DNA, the latter is a method referred to as "Quick change". PCR can also be used to determine whether a particular DNA fragment is found in a cDNA library. PCR has many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA, and, more recently, real-time PCR (QPCR) which allow for quantitative measurement of DNA or RNA molecules.

Principle

The core principle behind microarrays is hybridization between two DNA strands, the property of complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs. A high number of complementary base pairs in a nucleotide sequence means tighter non-covalent bonding between the two strands. After washing off of non-specific bonding sequences, only strongly paired strands will remain hybridized. So fluorescently labelled target sequences that bind to a probe sequence generate a signal that depends on the strength of the hybridization determined by the number of paired bases, the hybridization conditions (such as temperature), and washing after hybridization. Total strength of the signal, from a spot (feature), depends upon the amount of target sample binding to the probes present on that spot. Microarrays use relative quantitation in which the intensity of a feature is compared to the intensity of the same feature under a different condition, and the identity of the feature is known by its position.

Spotted vs. in situ synthesised arrays

Microarrays can be fabricated using a variety of technologies, including printing with fine-pointed pins onto glass slides, photolithography using pre-made masks, photolithography using dynamic micromirror devices, ink-jet printing, or electrochemistry on microelectrode arrays.

In spotted microarrays, the probes are oligonucleotides, cDNA or small fragments of PCR products that correspond to mRNAs. The probes are synthesized prior to deposition on the array surface and are then "spotted" onto glass. A common approach utilizes an array of fine pins or needles controlled by a robotic arm that is dipped into wells containing DNA probes and then depositing each probe at designated locations on the array surface. The resulting "grid" of probes represents the nucleic acid profiles of the prepared probes and is ready to receive complementary cDNA or cRNA "targets" derived from experimental or clinical samples. This technique is used by research scientists around the world to produce "in-house" printed microarrays from their own labs.

These arrays may be easily customized for each experiment, because researchers can choose the probes and printing locations on the arrays, synthesize the probes in their own lab (or collaborating facility), and spot the arrays. They can then generate their own labeled samples for hybridization, hybridize the samples to the array, and finally scan the arrays with their own equipment. This provides a relatively low-cost microarray that may be customized for each study, and avoids the costs of purchasing often more expensive commercial arrays that may represent vast numbers of genes that are not of interest to the investigator. Publications exist which indicate in-house spotted microarrays may not provide the same level of sensitivity compared to commercial oligonucleotide arrays, possibly owing to the small batch sizes and reduced printing efficiencies when compared to industrial manufactures of oligo arrays.

Two-channel vs. one-channel detection

Two-color microarrays or two-channel microarrays are typically hybridized with cDNA prepared from two samples to be compared (e.g. diseased tissue versus healthy tissue) and that are labeled with two different fluorophores.[Fluorescent dyes commonly used for cDNA labeling include Cy3, which has a fluorescence emission wavelength of 570 nm (corresponding to the green part of the light spectrum), and Cy5 with a fluorescence emission wavelength of 670 nm (corresponding to the red part of the light spectrum). The two Cy-labeled cDNA samples are mixed and hybridized to a single microarray that is then scanned in a microarray scanner to visualize fluorescence of the two fluorophores after excitation with a laser beam of a defined wavelength. Relative intensities of each fluorophore may then be used in ratio-based analysis to identify up-regulated and down-regulated genes.

Oligonucleotide microarrays often carry control probes designed to hybridize with RNA spike-ins. The degree of hybridization between the spike-ins and the control probes is used to normalize the hybridization measurements for the target probes. Although absolute levels of gene expression may be determined in the two-color array in rare instances, the relative differences in expression among different spots within a sample and between samples is the preferred method of data analysis for the two-color system. Examples of providers for such microarrays includes Agilent with their Dual-Mode platform, Eppendorf with their DualChip platform for colorimetric Silverquant labeling, and TeleChem International with Arrayit.

Experimental Design

Due to the biological complexity of gene expression, the considerations of experimental design that are discussed in the expression profiling article are of critical importance if statistically and biologically valid conclusions are to be drawn from the data.

There are three main elements to consider when designing a microarray experiment. First, replication of the biological samples is essential for drawing conclusions from the experiment. Second, technical replicates (two RNA samples obtained from each experimental unit) help to ensure precision and allow for testing differences within treatment groups. The technical replicates may be two independent RNA extractions or two aliquots of the same extraction. Third, spots of each cDNA clone or oligonucleotide are present as replicates (at least duplicates) on the microarray slide, to provide a measure of technical precision in each hybridization. It is critical that information about the sample preparation and handling is discussed, in order to help identify the independent units in the experiment and to avoid inflated estimates of statistical significance.

Relation between probe and gene

The relation between a probe and the mRNA that it is expected to detect is problematic. On the one hand, some mRNAs may cross-hybridize probes in the array that are supposed to detect another mRNA. In addition, mRNAs may experience amplification bias that is sequence or molecule-specific. On the other hand, probes that are designed to detect the mRNA of a particular gene may be relying on genomic EST information that is incorrectly associated with that gene.