Suspension array technology

Last updated November 24, 2022

Suspension array technology (or SAT) is a high throughput, large-scale, and multiplexed screening platform used in molecular biology. SAT has been widely applied to genomic and proteomic research, such as single nucleotide polymorphism (SNP) genotyping, genetic disease screening, gene expression profiling, screening drug discovery and clinical diagnosis.^[1]^[2]^[3] SAT uses microsphere beads (5.6 um in diameter) to prepare arrays. SAT allows for the simultaneous testing of multiple gene variants through the use of these microsphere beads as each type of microsphere bead has a unique identification based on variations in optical properties, most common is fluorescent colour. As each colour and intensity of colour has a unique wavelength, beads can easily be differentiated based on their wavelength intensity. Microspheres are readily suspendable in solution and exhibit favorable kinetics during an assay. Similar to flat microarrays (e.g. DNA microarray), an appropriate receptor molecule, such as DNA oligonucleotide probes, antibodies, or other proteins, attach themselves to the differently labeled microspheres. This produces thousands of microsphere array elements. Probe-target hybridization is usually detected by optically labeled targets, which determines the relative abundance of each target in the sample.^[4]

Overview of SAT using DNA hybridization

DNA is extracted from cells used to create test fragments. These test fragments are added to a solution containing a variety of microsphere beads. Each type of microsphere bead contains a known DNA probe with a unique fluorescent identity. Test fragments and probes on the microsphere beads are allowed to hybridize to each other. Once hybridized, the microsphere beads are sorted, usually using flow cytometry. This allows for the detection of each of the gene variants from the original sample. The resulting data collected will indicate the relative abundance of each hybridized sample to the microsphere.

Multiplexing

Since microsphere beads are easily suspended in solution and each microsphere retains its identity when hybridized to the test sample, a typical suspension array experiment can analyze a wide range of biological analysis in a single reaction, called "multiplexing". In general, each type of microsphere used in an array is individually prepared in bulk. For example, the commercially available microsphere arrays from Luminex xMAP technology uses a 10X10 element array. This array involves beads with red and infrared dyes, each with ten different intensities, to give a 100-element array.^[4] Thus, the array size would increase exponentially if multiple dyes are used. For example, five different dyes with 10 different intensities per dye will give rise to 100,000 different array elements.

Procedure

Sample targeting

When using different types of microspheres, SAT is capable of simultaneously testing multiple variables, such as DNA and proteins, in a given sample. This allows SAT to analyze variety of molecular targets during a single reaction. The common nucleic acid detection method includes direct DNA hybridization. The direct DNA hybridization approach is the simplest suspension array assay whereby 15 to 20 bp DNA oligonucleotides attached to microspheres are amplified using PCR. This is the optimized probe length as it minimizes the melting temperature variation among different probes during probe-target hybridization.^[1] After amplifying one DNA oligoprobe of interest, it can be used to create 100 different probes on 100 different sets of microspheres, each with the capability of capturing 100 potential targets (if using a 100-plex array). Similarly, target DNA samples are usually PCR amplified and labeled.^[4] Hybridization between the capture probe and the target DNA is achieved by melting and annealing complementary target DNA sequences to their capture probes located on the microspheres. After washing to remove non-specific binding between sequences, only strongly paired probe-targets will remain hybridized.^[1]

Sorting and detection with flow cytometry

For more details on this topic, see flow cytometry

Since the optical identity of each microsphere is known, the quantification of target samples hybridized to the microspheres can be achieved by comparing the relative intensity of target markers in one set of microspheres to target markers in another set of microspheres using flow cytometry. Microspheres can be sorted based using both their unique optical properties and level of hybridization to the target sequence.

Strengths

Rapid/high throughput: In multiplex analysis, a 100-plex assay can be analyzed in every 30 seconds. The recent reported high-throughput flow cytometry can sample a 96-well plate in 1 minute, and theoretically, the 100-plex assay with this system can be analyzed in less than 1 second, or potentially deliver 12 million samples per day.^[4]
High array density/multiplex: Compared to flat microarrays, SAT allows one to perform parallel measurements. A few microliters of microspheres could contain thousands of array elements and each array element is represented by hundreds of individual microspheres. Thus, the measurement by flow cytometry represents a replicate analysis of each array element.^[4]
Effective gathering of information: One of the benefits of using SAT is that it allows you to take one sample from a patient or research organism and simultaneously test for multiple gene variants. Thus, from a single sample you can determine which virus from a series of viruses a patient has, or which base pair mutation is present in the organism with a unique phenotype.^[3]
Cost-effective: Currently, commercially available suspension array kits costs $0.10-$0.25 per sequence tested.^[1]

Weaknesses

Relatively low array size: Although it has the potential to use an increased amount of dyes to generate millions of different array elements, the current generation of commercially available microsphere arrays (from Luminex xMAP technology) only uses two sets of dyes and therefore can only detect ~100 targets per experiment.^[4]
Hybridization between different sets of probes and target sequences requires a specific annealing temperature, which is affected by length and sequence of the oligonucleotide probe. Therefore, for every experiment, only one possible annealing temperature can be used. Thus, all probes used in given experiment must be designed to hybridize to the target at the same temperature. Although introducing base pair mismatch in some sets of the probes could minimize annealing temperature differences between each set of probes, the hybridization problem is still significant if more than 10-20 targets are tested in one reaction.^[1]

Related Research Articles

A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot contains picomoles of a specific DNA sequence, known as probes. These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA sample 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. The original nucleic acid arrays were macro arrays approximately 9 cm × 12 cm and the first computerized image based analysis was published in 1981. It was invented by Patrick O. Brown. An example of its application is in SNPs arrays for polymorphisms in cardiovascular diseases, cancer, pathogens and GWAS analysis. It is also used for the identification of structural variations and the measurement of gene expression.

<span class="mw-page-title-main">Flow cytometry</span> Lab technique in biology and chemistry

Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.

Comparative genomic hybridization(CGH) is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells. The aim of this technique is to quickly and efficiently compare two genomic DNA samples arising from two sources, which are most often closely related, because it is suspected that they contain differences in terms of either gains or losses of either whole chromosomes or subchromosomal regions. This technique was originally developed for the evaluation of the differences between the chromosomal complements of solid tumor and normal tissue, and has an improved resolution of 5–10 megabases compared to the more traditional cytogenetic analysis techniques of giemsa banding and fluorescence in situ hybridization (FISH) which are limited by the resolution of the microscope utilized.

Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific DNA sequences on chromosomes. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets in cells, circulating tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.

Molecular beacons, or molecular beacon probes, are oligonucleotide hybridization probes that can report the presence of specific nucleic acids in homogenous solutions. Molecular beacons are hairpin-shaped molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid sequence. This is a novel non-radioactive method for detecting specific sequences of nucleic acids. They are useful in situations where it is either not possible or desirable to isolate the probe-target hybrids from an excess of the hybridization probes.

In biology, a branched DNA assay is a signal amplification assay that is used to detect nucleic acid molecules.

<span class="mw-page-title-main">RNA spike-in</span>

An RNA spike-in is an RNA transcript of known sequence and quantity used to calibrate measurements in RNA hybridization assays, such as DNA microarray experiments, RT-qPCR, and RNA-Seq.

SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species. It is a form of genotyping, which is the measurement of more general genetic variation. SNPs are one of the most common types of genetic variation. An SNP is a single base pair mutation at a specific locus, usually consisting of two alleles. SNPs are found to be involved in the etiology of many human diseases and are becoming of particular interest in pharmacogenetics. Because SNPs are conserved during evolution, they have been proposed as markers for use in quantitative trait loci (QTL) analysis and in association studies in place of microsatellites. The use of SNPs is being extended in the HapMap project, which aims to provide the minimal set of SNPs needed to genotype the human genome. SNPs can also provide a genetic fingerprint for use in identity testing. The increase of interest in SNPs has been reflected by the furious development of a diverse range of SNP genotyping methods.

Multiplex ligation-dependent probe amplification (MLPA) is a variation of the multiplex polymerase chain reaction that permits amplification of multiple targets with only a single primer pair. It detects copy number changes at the molecular level, and software programs are used for analysis. Identification of deletions or duplications can indicate pathogenic mutations, thus MLPA is an important diagnostic tool used in clinical pathology laboratories worldwide.

Autonomous Detection Systems (ADS), also called biohazard detection systems or autonomous pathogen detection systems, are designed to monitor air in an environment and to detect the presence of airborne chemicals, toxins, pathogens, or other biological agents capable of causing human illness or death. These systems monitor the air continuously and send real-time alerts to appropriate authorities in the event of an act of bioterrorism or biological warfare.

A nucleic acid test (NAT) is a technique used to detect a particular nucleic acid sequence and thus usually to detect and identify a particular species or subspecies of organism, often a virus or bacterium that acts as a pathogen in blood, tissue, urine, etc. NATs differ from other tests in that they detect genetic materials rather than antigens or antibodies. Detection of genetic materials allows an early diagnosis of a disease because the detection of antigens and/or antibodies requires time for them to start appearing in the bloodstream. Since the amount of a certain genetic material is usually very small, many NATs include a step that amplifies the genetic material—that is, makes many copies of it. Such NATs are called nucleic acid amplification tests (NAATs). There are several ways of amplification, including polymerase chain reaction (PCR), strand displacement assay (SDA), or transcription mediated assay (TMA).

2 Base Encoding, also called SOLiD, is a next-generation sequencing technology developed by Applied Biosystems and has been commercially available since 2008. These technologies generate hundreds of thousands of small sequence reads at one time. Well-known examples of such DNA sequencing methods include 454 pyrosequencing, the Solexa system and the SOLiD system. These methods have reduced the cost from $0.01/base in 2004 to nearly $0.0001/base in 2006 and increased the sequencing capacity from 1,000,000 bases/machine/day in 2004 to more than 100,000,000 bases/machine/day in 2006.

The Illumina Methylation Assay using the Infinium I platform uses 'BeadChip' technology to generate a comprehensive genome-wide profiling of human DNA methylation. Similar to bisulfite sequencing and pyrosequencing, this method quantifies methylation levels at various loci within the genome. This assay is used for methylation probes on the Illumina Infinium HumanMethylation27 BeadChip. Probes on the 27k array target regions of the human genome to measure methylation levels at 27,578 CpG dinucleotides in 14,495 genes. The Infinium HumanMethylation450 BeadChip array targets > 450,000 methylation sites.

<span class="mw-page-title-main">MAGIChip</span>

MAGIChips, also known as "microarrays of gel-immobilized compounds on a chip" or "three-dimensional DNA microarrays", are devices for molecular hybridization produced by immobilizing oligonucleotides, DNA, enzymes, antibodies, and other compounds on a photopolymerized micromatrix of polyacrylamide gel pads of 100x100x20µm or smaller size. This technology is used for analysis of nucleic acid hybridization, specific binding of DNA, and low-molecular weight compounds with proteins, and protein-protein interactions.

Multiplex polymerase chain reaction refers to the use of polymerase chain reaction to amplify several different DNA sequences simultaneously. This process amplifies DNA in samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler. The primer design for all primers pairs has to be optimized so that all primer pairs can work at the same annealing temperature during PCR.

Molecular Inversion Probe (MIP) belongs to the class of Capture by Circularization molecular techniques for performing genomic partitioning, a process through which one captures and enriches specific regions of the genome. Probes used in this technique are single stranded DNA molecules and, similar to other genomic partitioning techniques, contain sequences that are complementary to the target in the genome; these probes hybridize to and capture the genomic target. MIP stands unique from other genomic partitioning strategies in that MIP probes share the common design of two genomic target complementary segments separated by a linker region. With this design, when the probe hybridizes to the target, it undergoes an inversion in configuration and circularizes. Specifically, the two target complementary regions at the 5’ and 3’ ends of the probe become adjacent to one another while the internal linker region forms a free hanging loop. The technology has been used extensively in the HapMap project for large-scale SNP genotyping as well as for studying gene copy alterations and characteristics of specific genomic loci to identify biomarkers for different diseases such as cancer. Key strengths of the MIP technology include its high specificity to the target and its scalability for high-throughput, multiplexed analyses where tens of thousands of genomic loci are assayed simultaneously.

Quantitative Fluorescent in situ hybridization (Q-FISH) is a cytogenetic technique based on the traditional FISH methodology. In Q-FISH, the technique uses labelled synthetic DNA mimics called peptide nucleic acid (PNA) oligonucleotides to quantify target sequences in chromosomal DNA using fluorescent microscopy and analysis software. Q-FISH is most commonly used to study telomere length, which in vertebrates are repetitive hexameric sequences (TTAGGG) located at the distal end of chromosomes. Telomeres are necessary at chromosome ends to prevent DNA-damage responses as well as genome instability. To this day, the Q-FISH method continues to be utilized in the field of telomere research.

Luminex Corporation | A DiaSorin Company is a biotechnology company which develops, manufactures and markets proprietary biological testing technologies with applications in life-sciences.

Spatial transcriptomics is a method for assigning cell types to their locations in the histological sections. This method can also be used to determine subcellular localization of mRNA molecules. The term is a variation of Spatial Genomics, first described by Doyle, et al., in 2000 and then expanded upon by Ståhl et al. in a technique developed in 2016, which has since undergone a variety of improvements and modifications.

References

1 2 3 4 5 Dunbar, Sherry A. (2006). "Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection". Clinica Chimica Acta. 363 (1–2): 71–82. doi:10.1016/j.cccn.2005.06.023. PMC 7124242 . PMID 16102740.
↑ Seideman, Jonathan; Peritt, David (2002). "A novel monoclonal antibody screening method using Luminex-100 microsphere system". Journal of Immunological Methods. 267 (2): 165–171. doi:10.1016/s0022-1759(02)00168-0. PMID 12165438.
1 2 Dunbar, Sherry A.; Vander Zee, Coe A.; Oliver, Kerry G.; Karem, Kevin L.; Jacobson, James W. (2003). "Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system". Journal of Microbiological Methods. 53 (2): 245–252. doi:10.1016/S0167-7012(03)00028-9. PMID 12654495.
1 2 3 4 5 6 Nolan, John P.; Sklar, Larry A. (2002). "Suspension array technology: evolution of the flat-array paradigm". Trends in Biotechnology. 20 (1): 9–12. doi:10.1016/s0167-7799(01)01844-3. PMID 11742671.

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[rev-1] 1 2 3 4 5 Dunbar, Sherry A. (2006). "Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection". Clinica Chimica Acta. 363 (1–2): 71–82. doi:10.1016/j.cccn.2005.06.023. PMC 7124242 . PMID 16102740.

[2] Seideman, Jonathan; Peritt, David (2002). "A novel monoclonal antibody screening method using Luminex-100 microsphere system". Journal of Immunological Methods. 267 (2): 165–171. doi:10.1016/s0022-1759(02)00168-0. PMID 12165438.

[bac-3] 1 2 Dunbar, Sherry A.; Vander Zee, Coe A.; Oliver, Kerry G.; Karem, Kevin L.; Jacobson, James W. (2003). "Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system". Journal of Microbiological Methods. 53 (2): 245–252. doi:10.1016/S0167-7012(03)00028-9. PMID 12654495.

[SAT-4] 1 2 3 4 5 6 Nolan, John P.; Sklar, Larry A. (2002). "Suspension array technology: evolution of the flat-array paradigm". Trends in Biotechnology. 20 (1): 9–12. doi:10.1016/s0167-7799(01)01844-3. PMID 11742671.

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