Suspension array technology (or SAT) is a high throughput, large-scale, and multiplexed screening platform used in
molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and phys ...
. SAT has been widely applied to
genomic
Genomics is an interdisciplinary field of biology focusing on the structure, function, evolution, mapping, and editing of genomes. A genome is an organism's complete set of DNA, including all of its genes as well as its hierarchical, three-dim ...
and
proteomic
Proteomics is the large-scale study of proteins. Proteins are vital parts of living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In ...
research, such as
single nucleotide polymorphism (SNP) genotyping,
genetic disease
A genetic disorder is a health problem caused by one or more abnormalities in the genome. It can be caused by a mutation in a single gene (monogenic) or multiple genes (polygenic) or by a chromosomal abnormality. Although polygenic disorders ...
screening,
gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. ...
profiling, screening drug discovery and clinical diagnosis.
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
A DNA microarray (also commonly known as DNA chip or biochip) 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 ...
), an appropriate receptor molecule, such as DNA
oligonucleotide
Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids ...
probes,
antibodies, or other
proteins
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respondi ...
, 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.
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
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, th ...
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
Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.
In this process, a sample containing cells or particles is suspended in a fluid and injected into the fl ...
. 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.
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
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respondi ...
, in a given sample. This allows SAT to analyze variety of molecular targets during a single reaction. The common
nucleic acid
Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main ...
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 PCR or pcr may refer to:
Science
* Phosphocreatine, a phosphorylated creatine molecule
* Principal component regression, a statistical technique
Medicine
* Polymerase chain reaction
** COVID-19 testing, often performed using the polymerase chain r ...
. This is the optimized probe length as it minimizes the melting temperature variation among different probes during probe-target hybridization.
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 PCR or pcr may refer to:
Science
* Phosphocreatine, a phosphorylated creatine molecule
* Principal component regression, a statistical technique
Medicine
* Polymerase chain reaction
** COVID-19 testing, often performed using the polymerase chain r ...
amplified and labeled.
Hybridization between the capture probe and the target
DNA is achieved by melting and annealing
complementary
A complement is something that completes something else.
Complement may refer specifically to:
The arts
* Complement (music), an interval that, when added to another, spans an octave
** Aggregate complementation, the separation of pitch-class ...
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.
Sorting and detection with flow cytometry
''For more details on this topic, see
flow cytometry
Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.
In this process, a sample containing cells or particles is suspended in a fluid and injected into the fl ...
''
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
Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.
In this process, a sample containing cells or particles is suspended in a fluid and injected into the fl ...
. 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
Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.
In this process, a sample containing cells or particles is suspended in a fluid and injected into the fl ...
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.
* 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
Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.
In this process, a sample containing cells or particles is suspended in a fluid and injected into the fl ...
represents a replicate analysis of each array element.
* 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.
* Cost-effective: Currently, commercially available suspension array kits costs $0.10-$0.25 per sequence tested.
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.
* 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.
References
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External links
Luminex products
Gene expression
Bioinformatics
DNA
Microtechnology
Molecular biology techniques
Microarrays