The Characteristics Of DNA Microarray Technology

Introduction

Humans have tens of thousands of genes, and the development of DNA microarrays by Patrick O. Brown, Joseph DeRisi, David Botstein, and colleagues in the mid-1990s made it possible to examine the expression of thousands of genes at once. Initial experiments using microarrays focused on determining which genes were expressed differently between normal cells and cancerous cells.

Over time, these methods have provided even more detail for physicians. For instance, microarrays are currently a key tool in genetic diagnosis, allowing doctors to identify specific subtypes within an overall disease category based on differences in gene expression. Moreover, physicians can use information from microarrays to determine which treatment methods will most likely yield beneficial results for particular cancer patients. But how do microarrays work, including its use in treatment of multiple mutation sheds light on both of these questions.

Save your time!
We can take care of your essay

• Proper editing and formatting
• Free revision, title page, and bibliography
• Flexible prices and money-back guarantee

Place an order

Scientists know that a mutation - or alteration - in a particular gene's DNA may contribute to a certain disease. it can be very difficult to develop a test to detect these mutations, because most large genes have many regions where mutations can occur.

DISCUSSION

The DNA microarray is a tool used to determine whether the DNA from a particular individual contains a mutation in genes the chip consists of a small glass plate encased in plastic. On the surface, each chip contains thousands of short,synthetic, single-stranded DNA sequences, which together add up to the normal gene in question, and to variants (mutations) of that gene that have been found in the human population.

DNA microarrays were used only as a research tool. Scientists continue today to conduct large-scale population studies this has become possible because, just as is the case for computer chips, very large numbers of 'features' can be put on microarray chips, representing a very large portion of the human genome.

Microarrays can also be used to study the extent to which certain genes are turned on or off in cells and tissues.

The DNA Microarray

This microarray scan has 90 spots arranged in 10 columns with 9 rows. The spots look like O’s and are red, green, and yellow against a black background.

DNA microarrays exploit the ability of complementary strands of nucleic acids to base-pair with each other and bind. For example, ATATGCGC will bind to its complement (TATACGCG) with a certain affinity.

This method was first used by Sol Spiegelman to measure the homology (similarity) of two different nucleic acids; Spiegelman called the method 'hybridization' of nucleic acids.

Later, the developers of the DNA microarray dotted an array of DNA copies (cDNAs) corresponding to a large number of different mRNAs of known sequence onto a glass slide. Because this array was so tiny, it was termed a microarray. Although the cDNAs were double-stranded, they could be melted, or denatured, to single strands, which could then be used to bind, or hybridize, to fluorescently labeled nucleic acid samples from cancerous or normal cells. After washing away the unbound molecules, bound fluorescent nucleic acid samples were identified by laser microscopy.

Fluorescent dots indicated expressed genes, and differences in microarray patterns between normal and cancerous cells could be quickly identified.

In these early microarray experiments, mRNA from one cell type was made into cDNA labeled with a red fluorescent dye, and mRNA from another cell type was made into cDNA labeled with a green fluorescent dye. The two cDNAs were then mixed and hybridized to the same DNA microarray, resulting in red, green, and yellow dots (caused by a combination of red and green).

The microarray is scanned to measure the expression of each gene printed on the slide. If the expression of a particular gene is higher in the experimental sample than in the reference sample, then the corresponding spot on the microarray appears red. In contrast, if the expression in the experimental sample is lower than in the reference sample, then the spot appears green. Finally, if there is equal expression in the two samples, then the spot appears yellow.

The data gathered through microarrays can be used to create gene expression profiles, which show simultaneous changes in the expression of many genes in response to a particular condition or treatment.

Comparative gene expression in the two samples could easily be determined by quantitating the ratio of red and green fluorescence in the spot corresponding to each gene.

DNA Microarray measurement of gene expression

A basic protocol for a DNA microarray is as follows: Isolate and purify mRNA from samples of interest. Since we are interested in comparing gene expression, one sample usually serves as control, and another sample would be the experiment (healthy vs. disease, etc)

RNA can be extracted from tissue or cell samples by common or­ganic extraction procedures used in most molecular biology labs. Both to­tal RNA and mRNA can be used for labelling.

The amount of to­tal RNA necessary for a single label­ling reaction is about 20 µg while the amount of mRNA necessary is about 0.5 µg. Lesser amounts are known to work, but require extreme purity and well developed protocols.

Reverse transcribe and label the mRNA. In order to detect the transcripts by hybridization, they need to be labeled, and because starting material maybe limited, an amplification step is also used. Labeling usually involves performing a reverse transcription (RT) reaction to produce a complementary DNA strand (cDNA) and incorporating a florescent dye( most commonly Cy3 and Cy5.) that has been linked to a DNA nucleotide, producing a fluorescent cDNA strand.

Disease and healthy samples can be labeled with different dyes and cohybridized onto the same microarray in the following step. Some protocols do not label the cDNA but use a second step of amplification, where the cDNA from RT step serves as a template to produce a labeled cRNA strand.

Purification of the Labelled Products. Once fluorescently labelled probes are made, the free unincorporated nucleotides must be removed. This is typically done by column chro­matography using convenient spin-columns or by ethanol precipitation of the sample.Some protocols perform both purification steps.

Hybridize the labeled target to the microarray. This step involves placing labeled cDNAs onto a DNA microarray where it will hybridize to their synthetic complementary DNA probes attached on the microarray. A series of washes are used to remove non-bound sequences.

Scan the microarray and quantitate the signal. The fluorescent tags on bound cDNA are excited by a laser and the fluorescently labeled target sequences that bind to a probe generate a signal.

The total strength of the signal depends upon the amount of target sample binding to the probes present on that spot. Thus, the amount of target sequence bound to each probe correlates to the expression level of various genes expressed in the sample. The signals are detected, quantified, and used to create a digital image of the array.

There are three basic types of microarrays: (A) Spotted array on glass. (B) Self assembles array. (C) in-situation synthesized array.

B. Self assembled arrays can be created by applying a collection of beads containing a diverse set of oligos to a surface with pits the size of the beads. After the array is constructed a series of hybridizations determine which oligo is in what position on each unique array.

C1 and C2. In-situ synthesized arrays can be produced by inkjet oligo synthesis methods (C1) or by photolithographic methods such as used by Affymetrix (C2).

Spotted arrays In 1996 Derisi et. al. published a method which allowed very high-density DNA arrays to be made on glass substrates(DeRisi et al., 1996). Poly-lysine coated glass microscope slides provided good binding of DNA and a robotic spotter was designed to spot multiple glass slide arrays from DNA stored in microtiter dishes. By using slotted pins (similar to fountain pens in design) a single dip of a pin in DNA solution could spot multiple slides. Spotting onto glass, allowed one to fluorescently label the sample. Fluorescent detection provided several advantages relative to the radioactive or chemiluminescent labels common to filter based arrays. First, fluorescent detection is quite sensitive and has a fairly large dynamic range. Second, fluorescent labeling is generally less expensive and less complicated than radioactive or chemiluminescent labeling. Third, fluorescent labeling allowed one to label two (or potentially more) samples in different colors and cohybridize the samples to the same array. As it was very difficult to reproducibly produce spotted arrays, comparisons of individually hybridized samples to ostensibly identical arrays would result in false differences due to array-to-array variation. However, a two-color approach in which the ratio of signals on the same array are measured is much more reproducible.

In-situ, Synthesized arrays In 1991 Fodor et.al. published a method for light directed, spatially addressable chemical synthesis which combined photolabile protecting groups with photolithography to perform chemical synthesis on a solid substrate(Fodor et al., 1991). In this initial work, the authors demonstrated the production of arrays of 10-amino acid peptides and, separately, arrays of di-nucleotides. In 1994, Fodor et.al. at the recently formed company of Affymetrix demonstrated the ability to use this technology to generate DNA arrays consisting of 256 different octa-nucleotides (Pease et al., 1994). By 1995-1996, Affymetrix arrays were being used to detect mutations in the reverse transcriptase and protease genes of the highly polymorphic HIV-1 genome(Lipshutz et al., 1995) and to measure variation in the human mitochondrial genome(Chee et al., 1996). Eventually, Affymetrix used this technology to develop a wide catalogue of DNA arrays for use in expression analysis(Lockhart et al., 1996; Wodicka et al., 1997), genotyping (Chee et al., 1996; Hacia et al., 1996) and sequencing (G Wallraff, 1997)(see www.Affymetrix.com for the current catalog of arrays).

A major advantage of the Affymetrix technology is that because the DNA sequences are directly synthesized on the surface, only a small collection of reagents (the 4 modified nucleotides, plus a small handful of reagents necessary for the de-blocking and coupling steps) are needed to construct an arbitrarily complex array. This contrasts with the spotted array technologies in which one needed to construct or obtain all the sequences that one wished to deposit on the array in advance of array construction. However, the initial Affymetrix technology was limited in flexibility as each model of array required the construction of a unique set of photolithographic masks in order to direct the light to the array at each step of the synthesis process. In 2002, authors from Nimblegen Systems Inc., published a method in which the photo-deprotection step of Fodor et. al (Fodor et al., 1991; Lipshutz et al., 1999) is accomplished using micro-mirrors (similar to those in video computer projectors) to direct light at the pixels on the array(Nuwaysir et al., 2002). This allows for custom arrays to be manufactured in small volumes at much lower cost than by photolithographic methods using masks to direct light (which are cheaper for large volume production). One constraint with this method is that the total number of addressable pixels (e.g. unit oligos that can be synthesized) is limited to the number of addressable positions in the micro-mirror device (of order 1M).

In 1996, Blanchard et.al. proposed a method use inkjet printing technology and standard oligo synthesis chemistry to produce oligo arrays(Blanchard et al., 1996). In brief, inkjet printer heads were adapted to deliver to the four different nucleotide phosphoramidites to a glass slide that was pre-patterned to contain regions containing hydrophilic regions (with exposed hydroxyl groups) surrounded by hydrophobic regions. The hydroxylated regions provided a surface to which the phosphoramidites could couple, while the surrounding hydrophobic regions contained the droplet(s) emitted by the inkjets to defined regions. This technology was eventually commercialized by Rosetta Inpharmatics (Hughes et al., 2001) and licensed to Agilent Technologies who produces these arrays at present. The inkjet array approach shares the advantage of the Affymetrix/Nimblegen approach in that one only need to have available a small number of reagents to produce an array. In addition, similar to the Nimblegen approach, the production of a new type of array only requires that a different set of sequence information is delivered to the printer. Hence, the inkjet array technology has been particularly useful for the design of custom arrays that are produced in low volume.

Self assembled arrays An alternative approach to the construction of arrays was created by the group of David Walt at Tufts University(Ferguson et al., 2000; Michael et al., 1998; Steemers et al., 2000; Walt, 2000) and ultimately licensed to Illumina. Their method involved synthesizing DNA on small polystryrene beads and depositing those beads on the end of a fiber optic array in which the ends of the fibers were etched to provide a well that is slightly larger than one bead. Different types of DNA would be synthesized on different beads and applying a mixture of beads to the fiber optic cable would result in a randomly assembled array. In early versions of these arrays, the beads were optically encoded with different fluorophore combinations in order to allow one to determine which oligo was in which position on the array (referred to as “decoding the array”)(Ferguson et al., 2000; Michael et al., 1998; Steemers et al., 2000; Walt, 2000). Optical decoding by fluorescent labeling limited the total number of unique beads that could be distinguished. Hence, the later and present day methods for decoding the beads involve hybridizing and detecting a number of short, fluorescently labeled oligos in a sequential series of steps(Gunderson et al., 2004). This not only allows for an extremely large number of different types of beads to be used on a single array but also functionally tests the array prior to its use in a biological assay. Later versions of the Illumina arrays used a pitted glass surface to contain the beads instead of a fiber option arrays.

The above is not intended to be a comprehensive history or survey of all DNA microarray technologies. However, it does cover the major advances in the field and the predominate methods of manufacture of arrays.

References

1. Roger Bumgamer, DNA microarrays: Types, application and their future, Published 2014 May 6, Mol. Biol. 101:22.1.1–22.1.11.
2. Victor Trevino, Francesco Falciani, and Hugo A.Barrera-Saldañia DNA microarrays: A powerful genomic tool for biomedical and clinical research, Mol Med. 2007 Sep-Oct; 13(9-10): 527–541.
3. Rajeshwar Govindarajan, Jeyapradha Duraiyan, Karunakaran Kaliyappan and Murugesan Palanisamy, Microarray and it’s applications, J Pharm Bioallied Sci. 2012 Aug; 4(Suppl 2): S310–S312.