Approaches of DNA Microarrays

February 14, 2024 Gaurab Karki Molecular Biology 0




Approaches of DNA Microarrays

Introduction:

  • DNA Microarray is one of the molecular detection techniques which is a collection of microscopic characteristics (commonly DNA) affixed to a solid surface.
  • Also termed as DNA chip or biochip.
  • DNA spots can be probed with the target molecules to result either qualitative or quantitative data.
  • Microarrays can be characterized on the basis of the nature of the probe, the solid support used and the specific technique used for target detection and/or probe addressing

Approaches of DNA microarrays

Some of the approaches of microarrays are;

  1. Printed microarrays
  2. In-situ synthesized microarrays
  3. High-density bead arrays
  4. Electronic microarrays
  5. Suspension bead microarrays

1. Printed Microarrays:

  • These were first microarrays to be used in research laboratories.
  • It is termed as ‘printed microarrays’ because of the printing or spotting of probes on the microarray surface (generally a glass slide).
  • The probe spots can be put in either by contact or noncontact printing.
  • The direct application of probe solution onto the microarray surface is performed by print pin in contact printing whereas the noncontact printer uses the same technology as the computer printers to eject the droplets of probe solution onto the glass slide.
  • Few nanolitres of probe solution per spot is applied to create an array of 100-150mm features, in both the cases of printed microarrays.
  • During the course of printing, it is important to control for cross-contamination and conserve the wholeness of the microarray and following hybridization data.
  • On account of comparatively large size of the characters, printed microarrays are lower in density(10,000-30,000 characters) than high density bead arrays and in situ-synthesized microarrays but provide more features than suspension bead arrays or electronic microarrays.
  • Printed are comparatively simple and economic than in situ  synthesized microarrays. However, the initial step of microarray facilities is expensive and requires separate space where environmental conditions such as humidity, dust, and temperature are strictly controlled.
  • A major merit of printed microarrays is flexibility. In clinical     microbiology, printed microarrays are used due to the ability to adjust spotted probes based upon revised annotations or the finding of noble, rising pathogens.
  • However, its use is convoluted by the tedious and expensive tasks of observing manufacture reproducibility, performing clinical validation studies, and continually evaluating the quality of downstream data.
  • Even if printed microarrays are favourable to user-defined testing, their use in diagnostic microbiology remains confined to specific research applications.

On the basis of nature of the probes, printed microarrays can be classified as:

1. Double stranded DNA microarrays:

  • The probes contains of amplicons acquired by PCR using the primers designed either from a familiar genomic sequence, or cDNA or shot-gun library clones.
  • The denaturation of the double stranded amplicons takes place either in print buffer or after immobilization, permitting the probes to be available for hybridization.
  • The attachment of the amplicons to the glass slide is favoured by the electrostatic interaction of the negative charge bore by the phosphate backbone of the DNA with the positive charge of the coating of the glass slide surface. It can also take place by the formation of UV-cross-linked covalent bonds between the thymidine bases in the DNA and amine groups on slides.
  • PCR amplicons are ideally required to have high specificity and yield eliminating contamination, including nonspecific amplification and taints that affect attachment to the microarray surface. But, dsDNA probes have high sensitivity but lacks in specificity. Less specificity can be advantageous when analyzing a genomic sequence rich in natural polymorphisms.
  • A great demerit in the production of printed dsDNA microarray is the massive scale of amplicon production and related troubles of quality control, information management, efficiency and preciseness.

2. Oligo-nucleotide microarrays:

  • In this type, the spotted probes contains of short, chemically synthesized sequences.
  • The probe’s length ranges from 25 to 80bp but it may be as long as 150bp for gene expression microarrays.
  • Shorter probe lengths allows less errors during probe synthesis and enables the interrogation of small genomic regions, plus polymorphisms. Specificity is greater when shorter and specific genomic regions are interrogated. With increasing length of the probe, the strength of the hybridization signal and the sensitivity increases.
  • In order to improve the hybridization signal strength, the higher concentration of the probes can be applied during printing.
  • Despite being easier to produce than dsDNA probes, oligonucleotide probes need to be carefully designed so that all probes acquire similar melting temperatures (within 50 C) and eliminate palindromic sequences.
  • The probe’s attachment to the glass slides takes place by the covalent linkage as electrostatic immobilization and cross-linking can result in significant loss of probes during wash steps due to their small size. The coupling of probes to the microarray surface takes place via modified 5′ to 3′ ends on coated slides that provide functional groups (epoxy or aldehyde)

2. In-situ synthesized Arrays:

  • In-situ synthesized arrays are immensely-high-density microarrays which use oligonucleotide probes, (out of which Gene chips are most extensively known).
  • Contrasting to printed oligonucleotide array, the oligonucleotide probes which is typically 1-2 cm2 are synthesized directly on the surface of the microarray.
  • As in-situ synthesized probes are usually short(20-25 bp), multiple probes per target are used to enhance statistical accuracy, sensitivity and specificity.
  • Normally,11 probes are used per 600 bases being examined.
  • The specificity is further increased by use of probe sets. A probe set consists of one perfect-match probe and one mismatch probe that contains a 1bp difference in middle position of probe. Each member of probe set is present in a separate feature, allowing the mismatch probe to behave as a negative control to recognize possible nonspecific cross-hybridization events.
  • Recent progresses in Gene Chips consists the use of longer probes, the design of arrays that cross-examine across entire genes or exons, and the execution of multiple self-standing and non-overlapping perfect-match probes in place of classic probe sets.
  • Affymetrix Gene Chips classically have >106  characters per microarray depending on the inter-character distance. The probes are produced using semiconductor based photochemical synthesis.
  • Synthesis linkers reformed with light-sensitive protecting groups are available on the quartz surface. Thus, the microarray surface is chemically secured from a nucleotide addition.
  • When the array surface is exposed to UV light, reactive nucleotides revised with a photolabile protecting group can be joined to increasing oligonucleotide chains.
  • Photolithographic masks are used to target specific nucleotides to specific probe sites. Each mask has a designated pattern of windows, which acts as a filter to either transmit or obstruct UV light from specific characters on the chemically preserved microarray surface.
  • The areas of the microarray surface that has been exposed to the light will be deprotected and specific nucleotides can be added. The pattern of windows in each mask controls the sequence of nucleotide addition.
  • In-situ probe synthesis is thus completed through the cycling of masking, exposure to light, and the addition of either A,T,C or G bases to the growing oligonucleotide.
  • Further high density oligonucleotide arrays include those synthesized by Roche NimbleGen and Agilent technologies. These platforms use longer oligonucleotide probes(60-100bp), but NimbleGen and Agilent uses maskless photo-mediated manufacture, and inkjet technology respectively for the synthesis of probes.
  • Also, NimbleGen and Agilent platforms allow multi-color hybridization whereas experiments performed with GeneChips are confined to one label.
  • Synthesized microarrays relies on commercial synthesis due to complicated nature of chemical synthesis and huge expense required in production and are therefore favourable for user-defined progress.
  • The major merits to these systems are the duplicatibility of the production process and the standardization of reagents, instrumentation, and data analysis, all of which are applied to the clinical laboratory.
  • Regardless printed or synthesized, oligonucleotide arrays normally permits much cleaner downstream hybridization than amplicon based microarrays.
  • Although in-situ synthesized oligonucleotide microarrays are very vigorous systems and have important control measures included, there are recently none with direct diagnostic contagious disease applications that are commercially accessible.

3. High-density bead arrays:

  • Bead arrays issues a patterned substrate for the high-density spotting of target nuclei acids alike the printed and in situ-hybridized microarrays.
  • Bead arrays rely on 3-mm silica beads that haphazardly self gather onto one of two available substrates: the Sentrix Array Matrix (SAM) or the Sentrix Bead Chip.
  • The SAM comprises 96 1.4-mm fibre-optic bundles. Each bundle is a single array containing of 50,000 5-mm light conducting fibers, each of which is chemically engraved to form a microwell for a single bead.
  • Upto 1,536 types of bead gather onto each fibre bundle, resulting in 30 beads of each type in the array in case of universal bead array.
  • Each SAM permits the inquiry of 96 independent samples.
  • Bead chips are more suitable for high density applications such as whole-genome typing.
  • Contrast to the known locations of printed and in situ hybridized microarray characters, the beads in Bead arrays haphazardly group to their final location on the array.
  • Thus the bead location is mapped which is completed by a decoding process.
  • The SAM can be treated using a standard microtiter plate, which makes it manageable to standard automation and high-throughput processing.
  • The distance between individual arrays on the 16-sample Bead Chip is similar to that of a standard multichannel pipettor, thereby mediating ease of use.
  • Bead arrays can support up to 105 to 106 characters and have built-in repetition.
  • Since each manufactured microarray will not be similar, this redundancy is a pivotal experimental control for inter microarray comparative data.
  • An additional merit to the peculiarity of each microarray is that altering the bead pattern produces a method to identify spatial bias.
  • Although the analysis tools are present for Bead array-specific data analysis, background rectification, and spatial trace recognition have been delaying behind those provided by other microarray manufacturers.
  • Bead arrays have been usually applied to DNA methylation studies, gene expression profiling , and SNP genotyping.

4. Electronic microarrays:

  • The printed and in situ-synthesized microarrays and Bead arrays explained earlier depend on passive transport for the hybridization of nucleic acids. Dissimilar to it, electronic microarrays utilize active hybridization via electric fields to control nucleic acid transport.
  • For the electronic addressing of nucleic acids, microelectronic cartridges use complementary metal oxide semiconductor technology.
  • Each Nano Chip cartridge consists of 12 connectors that control 400 individual test sites.
  • When a positive current is applied to one or more test sites on the microarray, negatively charged nucleic acids are transported to specific sites, or features.
  • The surface of the microarray consists  streptavidin, which forms streptavidin-biotin bonds once electronically addressed biotinylated probes meet their targeted location. The positive current is then eliminated from the active features, and new test sites can be operated by the targeted application of a positive current.
  • The microarray is set  for the application of fluorescently labeled target DNA, once the probes have been hybridized at distinct features.
  • Usually, target DNA passively hybridizes with the immobilized probes on the microarray but can also be concentrated electronically. Even though addressing the capture probe down first is the most normally used format, amplicon-down and sandwich assays have also been employed.
  • Regardless of the addressing format used, if hybridization occurs between the probe and the target DNA, fluorescent reporters will be present at the positive test, which will be detected when the electronic microarray is examined and analysed.
  • Electronic microarrays has many merits. For instance, since multiple probes, each with a discrete fluorophore, can be sequentially addressed to the same feature, multiplex detection can be completed at a single test site.
  • The flexibility of this platform permits nucleic acids from an individual sample to be hybridized to multiple test sites for the spotting of multiple targets, or nucleic acids from multiple samples can be examined on the same microarray cartridge, decreasing waste.

5. Suspension bead arrays:

  •  Dissimilar to the two-dimensional, or planar, arrays explained earlier, suspension bead arrays are three- dimensional arrays which are based on the utilization of microscopic polystyrene spheres (beads) as the solid support and flow cytometry for bead and target spotting.
  • Furthermore, they are discrete from the high-density Illumina Bead arrays discussed earlier, in which the beads are placed on fiber- optic strands or silicon slides.
  • Suspension-bead-based assays were initially described in 1977 and focused on the detection of antibodies and antigens.
  • Multiplexing was initially achieved by using variable-sized microsphere sets for the continuous detection of multiple antibodies. Currently, more vigorous multiplexing is completed using different microsphere sets based on color.
  • Red (658-nm radiation) and infra- red (712-nm radiation) fluorochromes are used at different concentrations to fill 5.6-mm microspheres.
  • Each bead of the 100-microsphere set has a different red-to-infrared ratio, and thus, each bead has a distinct spectral address.
  • Microspheres with a specific spectral address integrated to a specific probe are identical to a character in a planar microarray.
  • Once multiple individual microspheres have been incorporated to separate specific probes, a mixture of microspheres can be used to examine extracted and amplified nucleic acids.
  • Using a bench-top flow cytometer, the succeeding  detection of a fluorescent reporter that indicates probe-target DNA hybridization is completed.
  • An individual file microsphere suspension proceeds by two lasers. A 635-nm laser stimulates the red and infrared fluorochromes infused in the microspheres, which permits the classification of the bead and thus the identity of the probe-target being examined. A 532-nm laser thrills reporter fluorochromes such as R-phycoerythrin and Alexa 532 to determine any hybridization that takes place on the microsphere.
  • For nucleic acid detection by suspension bead arrays, several chemistries have been initiated that includes direct DNA hybridization, competitive DNA hybridization, and solution based chemistries with microsphere capture. In direct DNA hybridization, PCR amplicons hybridize directly to probe capture sequences placed on the microspheres.
  • Principally, a biotinylated primer used during amplification permits streptavidin–R-phycoerythrin to attach and label hybridized microspheres.
  • Competitive DNA hybridization employs unlabeled PCR amplicons and biotinylated competitor oligonucleotides. In difference to the direct hybridization method, competitive DNA hybridization results high fluorescence in the lack of target DNA. When target DNA is available, it binds the tagged competitor DNA, which, in turn, is not present to hybridize to the microsphere, resulting low fluorescence.
  • Allele-specific primer extension (ASPE) or target-specific primer extension (TSPE), oligonucleotide ligation assay (OLA), and single-base-chain extension (SBCE) are solution-based chemistries integrated with succeeding microsphere capture.
  • By utilizing the natural properties of DNA polymerases and ligases, these chemistries integrate a capture sequence during the solution-based reaction.
  • Both ASPE or TSPE and OLA use a capture primer, which consists a unique 5′ sequence succeeded  by a target-specific sequence. In ASPE and/or TSPE, the primer can be extended by DNA polymerase only if target DNA is available to supply the complementary base for the 3′ nucleotide. The tag in ASPE and/or TSPE is facilitated by a biotinylated deoxynucleotide triphosphate. The OLA reaction is ligase dependent. In addition to the capture primer, a biotinylated probe homologous to target DNA is present during an OLA.
  • The capture primer and reporter probes can be joined only if target DNA is present in the sample. Used for multiplex SNP detection, SBCE needs independent reactions for each nucleotide query. For every SNP being cross-examined, one probe with a distinct capture sequence is used to assay the possible alleles in separate wells consisting a distinct dideoxynucleoside triphosphate per well. When the capture and target sequences are homologous, a biotinylated dideoxynucleoside triphosphate is integrated, thereby stopping further augmentation.
  • The solution-based chemistries explained above all take advantage of universal microspheres with nonspecific capture sequences.
  • The first universal sequences used to label microspheres were ZipCode/cZipCode arrest sequences originally used with SBCE in SNP genotyping assays. The 25-bp ZipCode sequences are based on erratic genomic sequences from Mycobacterium tuberculosis.
  • A distinct ZipCode sequence is incorporated in the 5′ end of the capture probe used in the chemistries explained above, while microspheres are labeled with the complementary sequence.
  • Even though the character density of suspension bead arrays is the least of all the platforms assessed, merits abound that make this platform the most practical for clinical microbiology applications.
  • The presence of universal bead sets and their innate flexibility make the progress of user-defined applications practical and relatively economic. Although users must carefully affirm the positive fluorescent threshold for each analyte in the multiplex, user-defined bead-based assays allow experienced users a multitude of clinically relevant applications.
  • Significantly, in 2008, Luminex obtained FDA clearance for the first infectious-disease suspension bead array (xTAG RVP), which diagnose 12 respiratory viruses and subtypes. Although analyte-specific reagents (ASRs) also exist, the presence of FDA-cleared products is an important step in getting this technology into less-experienced detective microbiology laboratories.
  • However, many built clinical molecular microbiology laboratories depends greatly on real-time PCR, which has less contamination risks.
  • In distinction, the opening of postamplification tubes and the following pipetting steps in the workflow of suspension arrays increase the risk for intra- and inter run contamination. Careful deliberation should be taken to control contamination and the reestablishment of postamplification laboratory space in the era of real-time PCR.
  • Nevertheless, the relative clarity, strong multiplexing abilities, and cost effectiveness of suspension bead arrays make this platform the most luring for high-throughput nucleic acid diagnosis in clinical contagious disease diagnostics.

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