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SYBR Gold is an asymmetrical
cyanine dye
Cyanines, also referred to as tetramethylindo(di)-carbocyanines are a synthetic dye family belonging to the polymethine group. Although the name derives etymologically from terms for shades of blue, the cyanine family covers the electromagnetic ...
. It can be used as a stain for double-stranded DNA, single-stranded DNA, and RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
. SYBR Gold is the most sensitive fluorescent stain of the SYBR family of dyes for the detection of nucleic acids
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 clas ...
. The SYBR family of dyes is produced by Molecular Probes Inc., now owned by Thermo Fisher Scientific
Thermo Fisher Scientific Inc. is an American supplier of scientific instrumentation, reagents and consumables, and software services. Based in Waltham, Massachusetts, Thermo Fisher was formed through the merger of Thermo Electron and Fisher S ...
SYBR Gold is more sensitive than ethidium bromide
Ethidium bromide (or homidium bromide, chloride salt homidium chloride) is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. It i ...
, SYBR Green I
SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. The SYBR family of dyes is produced by Molecular Probes Inc., now owned by Thermo Fisher Scientific. SYBR Green I binds to DNA. The resulting DN ...
, and SYBR Green II for detecting various types of nucleic acids
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 clas ...
. SYBR Gold's superior sensitivity is due to the high fluorescence quantum yield The quantum yield (Φ) of a radiation-induced process is the number of times a specific event occurs per photon absorbed by the system.
Applications
Fluorescence spectroscopy
The fluorescence quantum yield is defined as the ratio of the numb ...
of the dye-nucleic acid complexes (~0.6-0.7) and the dye's large fluorescence
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, tha ...
enhancement upon binding to nucleic acids
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 clas ...
(~1000-fold). SYBR Gold can detect as little as 25 pg of DNA which makes it >10-fold more sensitive than ethidium bromide
Ethidium bromide (or homidium bromide, chloride salt homidium chloride) is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. It i ...
for detecting nucleic acids
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 clas ...
in denaturing urea
Urea, also known as carbamide, is an organic compound with chemical formula . This amide has two amino groups (–) joined by a carbonyl functional group (–C(=O)–). It is thus the simplest amide of carbamic acid.
Urea serves an important r ...
, gyoxal, and formaldehyde
Formaldehyde ( , ) (systematic name methanal) is a naturally occurring organic compound with the formula and structure . The pure compound is a pungent, colourless gas that polymerises spontaneously into paraformaldehyde (refer to section F ...
gels
A gel is a semi-solid that can have properties ranging from soft and weak to hard and tough. Gels are defined as a substantially dilute cross-linked system, which exhibits no flow when in the steady-state, although the liquid phase may still dif ...
, even with 300 nm transillumination
Transillumination is the technique of sample illumination by transmission of light through the sample. Transillumination is used in a variety of methods of imaging.
Microscopy
In microscopy transillumination refers to the illumination of a sample ...
.
Fluorescence properties
SYBR Gold has twofluorescence
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, tha ...
excitation maxima when bound to DNA, one centered at ~300 nm and one at ~495 nm, and a single emission maximum at ~537 nm. SYBR Gold exhibits improved DNA cloning
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word ''cloning'' refers to the fact that the metho ...
efficiency compared to others dyes
A dye is a colored substance that chemically bonds to the substrate to which it is being applied. This distinguishes dyes from pigments which do not chemically bind to the material they color. Dye is generally applied in an aqueous solution and ...
of the SYBR family because it can be excited with blue light transillumination
Transillumination is the technique of sample illumination by transmission of light through the sample. Transillumination is used in a variety of methods of imaging.
Microscopy
In microscopy transillumination refers to the illumination of a sample ...
, which does not cause DNA damage. The fluorescence
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, tha ...
intensity increases linearly with the number of SYBR Gold molecules bound to DNA up to dye concentrations of ~ 2.5 µM, where quenching
In materials science, quenching is the rapid cooling of a workpiece in water, oil, polymer, air, or other fluids to obtain certain material properties. A type of heat treating, quenching prevents undesired low-temperature processes, such as pha ...
and inner filter effects become relevant. For dye concentrations ≤ 2.5 µM the fluorescence
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, tha ...
intensity as a function of the DNA concentration is well described by a global binding model for dye
A dye is a colored substance that chemically bonds to the substrate to which it is being applied. This distinguishes dyes from pigments which do not chemically bind to the material they color. Dye is generally applied in an aqueous solution an ...
concentrations.
Binding to DNA
SYBR Gold is anintercalator
In chemistry, intercalation is the reversible inclusion or insertion of a molecule (or ion) into layered materials with layered structures. Examples are found in graphite and transition metal dichalcogenides.
:
Examples Graphite
One famous inter ...
. This means it binds to DNA by insertion of one dye
A dye is a colored substance that chemically bonds to the substrate to which it is being applied. This distinguishes dyes from pigments which do not chemically bind to the material they color. Dye is generally applied in an aqueous solution an ...
molecule between two planar bases / base pairs
A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...
of DNA. Single molecule magnetic tweezers assays reveal systematic lengthening and unwinding of DNA by 19.1º ± 0.7º per dye
A dye is a colored substance that chemically bonds to the substrate to which it is being applied. This distinguishes dyes from pigments which do not chemically bind to the material they color. Dye is generally applied in an aqueous solution an ...
molecule upon binding, consistent with intercalation. This is similar to related dyes
A dye is a colored substance that chemically bonds to the substrate to which it is being applied. This distinguishes dyes from pigments which do not chemically bind to the material they color. Dye is generally applied in an aqueous solution and ...
like SYBR Green I
SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. The SYBR family of dyes is produced by Molecular Probes Inc., now owned by Thermo Fisher Scientific. SYBR Green I binds to DNA. The resulting DN ...
. The dissociation constant
In chemistry, biochemistry, and pharmacology, a dissociation constant (K_D) is a specific type of equilibrium constant that measures the propensity of a larger object to separate (dissociate) reversibly into smaller components, as when a complex fa ...
– a measure to describe the binding affinity
In biochemistry and pharmacology, a ligand is a substance that forms a complex with a biomolecule to serve a biological purpose. The etymology stems from ''ligare'', which means 'to bind'. In protein-ligand binding, the ligand is usually a mol ...
of SYBR Gold to double-stranded DNA – is 0.27 ± 0.03 µM.
Uses
SYBR Gold is used in several areas ofmolecular 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 physi ...
and biochemistry
Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology and ...
. Its main use is to visualise DNA in electrophoresis
Electrophoresis, from Ancient Greek ἤλεκτρον (ḗlektron, "amber") and φόρησις (phórēsis, "the act of bearing"), is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric fie ...
, for example agarose
Agarose is a heteropolysaccharide, generally extracted from certain red seaweed. It is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Agarose is o ...
or polyacrylamide gels. SYBR Gold is able to penetrate thick and high percentage agarose gels rapidly, and even formaldehyde
Formaldehyde ( , ) (systematic name methanal) is a naturally occurring organic compound with the formula and structure . The pure compound is a pungent, colourless gas that polymerises spontaneously into paraformaldehyde (refer to section F ...
agarose gels do not require de-staining, due to the low intrinsic fluorescence
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, tha ...
of the unbound dye
A dye is a colored substance that chemically bonds to the substrate to which it is being applied. This distinguishes dyes from pigments which do not chemically bind to the material they color. Dye is generally applied in an aqueous solution an ...
. SYBR Gold can be readily removed from nucleic acids
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 clas ...
by ethanol precipitation Ethanol precipitation is a method used to purify and/or concentrate RNA, DNA, and polysaccharides such as pectin and xyloglucan from aqueous solutions by adding ethanol as an antisolvent.
DNA precipitation Theory
DNA is polar due to its highl ...
, leaving pure templates available for subsequent manipulation or analysis.
Also, SYBR Gold can also be used for labelling
Labelling or using a label is describing someone or something in a word or short phrase. For example, the label "criminal" may be used to describe someone who has broken a law. Labelling theory is a theory in sociology which ascribes labelling ...
of DNA within cells for 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 flo ...
and fluorescence microscopy
A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microsc ...
.
To preserve fluorescence
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, tha ...
activity, SYBR Gold should be protected from light
Light or visible light is electromagnetic radiation that can be perceived by the human eye. Visible light is usually defined as having wavelengths in the range of 400–700 nanometres (nm), corresponding to frequencies of 750–420 tera ...
as much as possible, particularly once it has been diluted for use.
Safety
The SYBR family of dyes is marketed as a replacement forethidium bromide
Ethidium bromide (or homidium bromide, chloride salt homidium chloride) is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. It i ...
, a potential human mutagen, as both safer to work with and free from the complex waste disposal issues of ethidium
Ethidium bromide (or homidium bromide, chloride salt homidium chloride) is an intercalating agent commonly used as a fluorescent tag ( nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. It ...
. But no data are available addressing the mutagenicity
In genetics, a mutagen is a physical or chemical agent that permanently changes genetic material, usually DNA, in an organism and thus increases the frequency of mutations above the natural background level. As many mutations can cause cancer in ...
or toxicity
Toxicity is the degree to which a chemical substance or a particular mixture of substances can damage an organism. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a subst ...
of SYBR Gold. Because SYBR Gold binds to DNA in an intercalative way with high affinity which makes it a possible carcinogen
A carcinogen is any substance, radionuclide, or radiation that promotes carcinogenesis (the formation of cancer). This may be due to the ability to damage the genome or to the disruption of cellular metabolic processes. Several radioactive substan ...
. Therefore, SYBR Gold should be handled with particular caution and solutions of SYBR Gold stain should be disposed of in accordance with local regulationsSimilar cyanine dyes
*SYBR Green I
SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. The SYBR family of dyes is produced by Molecular Probes Inc., now owned by Thermo Fisher Scientific. SYBR Green I binds to DNA. The resulting DN ...
* SYBR Green II
* Pico Green
* SYBR Safe
SYBR Safe is a cyanine dye used as a nucleic acid stain in molecular biology. SYBR Safe is one of a number of SYBR dyes made by the Life Technologies Corporation. SYBR Safe binds to DNA. The resulting DNA-dye-complex absorbs blue light (λmax = ...
* SYTOX Orange
* Thiazole Orange
Thiazole, or 1,3-thiazole, is a heterocyclic compound that contains both sulfur and nitrogen. The term 'thiazole' also refers to a large family of derivatives. Thiazole itself is a pale yellow liquid with a pyridine-like odor and the molecular for ...
References
{{reflist Cyanine dyes Staining dyes