Molecular Probes

Section 8.1 — Nucleic Acid Stains

Molecular Probes prepares the most extensive assortment of nucleic acid stains commercially available, many of which have been developed in our research laboratories. This section discusses the physical properties of the various classes of dyes listed below. The sections in Chapter 8 that follow discuss numerous applications of these dyes and our other reagents and technology for genomics research.

The four classes of Molecular Probes' proprietary cyanine dyes include:

• Premier dyes for ultrasensitive nucleic acid quantitation and gel staining (Table 8.1)
• The cell-impermeant TOTO, TO-PRO and SYTOX families of dyes (Table 8.2)
• The cell-permeant SYTO family of dyes (Table 8.3)
• Chemically reactive SYBR dyes that can be used to form bioconjugates (see below)

The three classes of classic nucleic acid stains (Table 8.4) include:

• Intercalating dyes, such as ethidium bromide and propidium iodide
• Minor-groove binders, such as DAPI and the Hoechst dyes
• Miscellaneous nucleic acid stains, including acridine orange, 7-AAD, LDS 751 and hydroxystilbamidine, with special properties

Properties of Cyanine Dyes

Over the years, Molecular Probes researchers have invented many nucleic acid–binding cyanine dye derivatives that share several unique and outstanding properties:

• High molar absorptivity, with extinction coefficients typically greater than 50,000 cm^-1M^-1 at visible wavelengths
• Very low intrinsic fluorescence, with quantum yields usually less than 0.01 when not bound to nucleic acids
• Large fluorescence enhancements (often over 1000-fold) upon binding to nucleic acids, with increases in quantum yields to as high as 0.9
• Moderate to very high affinity for nucleic acids, with little or no staining of other biopolymers

Representatives of this class of nucleic acid stains have fluorescence excitations and emissions that span the visible-light spectrum from blue to near-infrared (Figure 8.1) with additional absorption peaks in the UV, making them compatible with many different types of instrumentation. The cyanine dyes show differences in some physical characteristics — particularly differences in permeability to cell membranes and nucleic acid specificity — that allow their distribution into distinct classes. Those classes are discussed in detail in the following sections of this chapter.

Premier Cyanine Dyes for Ultrasensitive Nucleic Acid Detection and Quantitation

Several of our cyanine dyes give superior results in specific assays for the analysis of nucleic acids (Table 8.1). For these dyes, we have developed detailed and extensively tested protocols to facilitate reproducible, high-sensitivity results in these assays.

• The PicoGreen, OliGreen and RiboGreen quantitation reagents in Section 8.3 set a benchmark for the detection and quantitation of DNA, RNA and oligonucleotides in solution. These reagents offer extremely simple and rapid protocols as well as linear ranges that span up to four orders of magnitude in nucleic acid concentration.
• The SYBR Gold, SYBR Green I and SYBR Green II nucleic acid gel stains in Section 8.4 are ultrasensitive gel stains that surpass the sensitivity of ethidium bromide by more than an order of magnitude in nucleic acid detection.
• The SYBR Safe DNA gel stain (Section 8.4) is not only significantly less mutagenic than ethidium bromide, but SYBR Safe stain's detection sensitivity is comparable to that of ethidium bromide. The SYBR Safe stain showed no or very low mutagenic activity when tested by an independent, licensed testing laboratory, and it is not classified as hazardous waste under U.S. Federal regulations (Product Highlight: SYBR Safe DNA Gel Stain).
• SYBR DX DNA blot stain (S7550, Section 8.5) allows the direct detection of DNA on filter membranes after Southern transfer, with sensitivity equivalent to that achieved with silver-enhanced gold staining.
• The CyQUANT GR dye (C7026) in Section 15.4 reagent for quantitating cell proliferation can reliably detect the nucleic acids in as few as 50 cells.

Cell-Impermeant Cyanine Dimers: The TOTO Family of Dyes

The Patented cyanine dimer dyes listed in Table 8.2 are often referred to as the TOTO family of dyes. These dyes are symmetric dimers of cyanine dyes with exceptional sensitivity for nucleic acids. This sensitivity is due to a high affinity for nucleic acids, in combination with a very high fluorescence enhancement and quantum yield upon binding. The unique physical characteristics of these dyes and some illustrative applications are discussed below. Specific applications are discussed in later sections of this chapter.

Each of the cyanine dimer dyes is available separately (Dimeric Cyanine Nucleic Acid Stains). For researchers designing new applications, the Nucleic Acid Stains Dimer Sampler Kit (N7565, Dimeric Cyanine Nucleic Acid Stains) provides samples of eight spectrally distinct analogs of the dimeric cyanine dyes for testing (Table 8.2).

High Affinity for Nucleic Acids

Appropriately designed dimers of nucleic acid–binding dyes have nucleic acid–binding affinities that are several orders of magnitude greater than those of their parent compounds. For example, the intrinsic DNA binding affinity constants of ethidium bromide (E1305, E3565) and ethidium homodimer-1 (E1169) are reported to be 1.5 × 10⁵ and 2 × 10⁸ M^-1, respectively, in 0.2 M Na⁺. As a result, the dimeric cyanine dyes are among the highest-affinity fluorescent probes available for nucleic acid staining. For example, in the TOTO-1 dimeric cyanine dye (T3600), the positively charged side chains of the TO-PRO-1 monomeric cyanine dye (T3602, ) are covalently linked to form the TOTO-1 molecule, with four positive charges (). This linkage gives the TOTO-1 dye a greatly enhanced affinity for nucleic acids — more than 100 times greater than that of the TO-PRO-1 monomer. The TOTO-1 dye exhibits a higher affinity for double-stranded DNA (dsDNA) than even the ethidium homodimers and also binds to both single-stranded DNA (ssDNA) and RNA. The extraordinary stability of TOTO-1–nucleic acid complexes ensures that the dye–DNA association remains stable, even during electrophoresis (); thus, samples can be stained with nanomolar dye concentrations prior to electrophoresis, thereby reducing the hazards inherent in handling large volumes of ethidium bromide staining solutions. In contrast, the binding of thiazole orange — the parent compound of TOTO-1 and TO-PRO-1 — is rapidly reversible, limiting the dye's sensitivity and rendering its nucleic acid complex unstable to electrophoresis.

High Fluorescence Enhancements and High Quantum Yields upon Binding to Nucleic Acids

In addition to their superior binding properties, TOTO-1 dye and the other cyanine dimers are essentially nonfluorescent in the absence of nucleic acids and exhibit fluorescence enhancements upon DNA binding of 100- to 1000-fold, which compares favorably with the fluorescence enhancement of thiazole orange upon DNA binding (~3000-fold). Furthermore, the fluorescence quantum yields of the cyanine dimers bound to DNA are high (generally between 0.2 and 0.6), and their extinction coefficients are an order of magnitude greater than those of the ethidium homodimers. This sensitivity is sufficient for detecting single molecules of labeled nucleic acids by optical imaging () and flow cytometry (Section 8.4) and for tracking dye-labeled virus particles in microbial communities and aquatic systems by fluorescence microscopy. These dyes are generally considered to be cell impermeant, although their use to stain reticulocytes permeabilized by 5% DMSO has been reported.

Modifying the Dimers Creates Compounds with Different Spectral Characteristics

Simply by changing the aromatic rings and the number of carbon atoms linking the cyanine monomers, we were able to synthesize an extended series of these dyes with different spectral characteristics (Table 8.2). Chemical modifications produce dramatic shifts in the absorption and emission spectra and reduce the quantum yields of the bound dyes but cause little or no change in their high affinity for DNA. The names of the dyes reflect their basic structure and spectral characteristics. For example, YOYO-1 iodide (491/509) has one carbon atom bridging the aromatic rings of the oxacyanine dye and exhibits absorption/emission maxima of 491/509 nm when bound to dsDNA. The YOYO-3 dye (612/631) — which differs from the YOYO-1 dye only in the number of bridging carbon atoms — has absorption/emission maxima of 612/631 nm when bound to dsDNA. Fluorescence spectra for the POPO, BOBO, YOYO, TOTO, JOJO and LOLO dyes bound to dsDNA are shown in Figure 8.1. The spectra of these dyes at dye:base ratios of less than 1:1 are essentially the same for the corresponding dye–ssDNA and dye–RNA complexes. At higher dye:base ratios, however, ssDNA and RNA complexes of all of the monomethine ("-1") dyes of the TOTO series and TO-PRO series have red-shifted emissions, whereas corresponding complexes of the trimethine ("-3") analogs do not. Thus, the cyanine dimer family provides dyes with a broad range of spectral characteristics to match the output of almost any available excitation source. Some common light sources that match each dye are shown in Table 8.2.

Binding Modes of the Cyanine Dimers

The studies on cyanine dimer binding modes have focused on the YOYO-1 and TOTO-1 dyes. The YOYO-1 dye was found to exhibit at least two distinct binding modes. At low dye:base pair ratios, the binding mode appears to consist primarily of bis-intercalation. Each monomer unit intercalates between bases, with the benzazolium ring system sandwiched between the pyrimidines and the quinolinium ring between the purine rings, causing the helix to unwind. The distortion in the local DNA structure caused by YOYO-1 bis-intercalation has been observed by two-dimensional NMR spectroscopy. At high dye:base pair ratios, a second, less well characterized mode of external binding begins to contribute. Circular dichroism measurements also indicate a possible difference in the binding modes of the YOYO-1 dye to ssDNA and dsDNA. These data are consistent with our own results, including the observation that the fluorescence emission of the YOYO-1 dye complex with nucleic acids shifts to longer wavelengths at high dye:base ratios upon binding to single-stranded nucleic acids and that the salt, ethanol and sodium dodecyl sulfate (SDS) sensitivity of YOYO-1 dye binding to DNA is a function of the dye:base pair ratio.

The TOTO-1 dye is capable of bis-intercalation, although it reportedly interacts with dsDNA and ssDNA with similarly high affinity. NMR studies of TOTO-1 dye interactions with a double-stranded 8-mer indicate that TOTO-1 dye is a bis-intercalator, with the fluorophores intercalating between the bases and the linker region having interactions in the minor groove (Figure 8.6). Binding of the dye partially unwinds the DNA, distorting and elongating the helix. However, another study using fluorescence polarization measurements suggests that an external binding mode, where the dipole of the dye molecule is aligned with the DNA grooves, may be more important. The TOTO-1 dye reportedly exhibits some sequence selectivity for the site 5'-CTAG-3', although it will bind to almost any sequence in dsDNA. The TOTO-1 dye does not exhibit cooperative binding to DNA, suggesting that it may be a suitable dye for detecting nucleic acids in gels.

The binding modes of the other members of the TOTO dye series have also been partially characterized. Electrophoresis and fluorescence lifetime measurements have shown that the YOYO-3 dye also appears to intercalate into DNA. During application development, we have determined that staining of nucleic acids by the BOBO-1 and POPO-1 dyes is much faster (occurring within minutes) than staining by the YOYO-1 or TOTO-1 dyes (which can take several hours to reach equilibrium under the same experimental conditions), indicating possible differences in their binding mechanisms. Fluorescence yield and lifetime measurements have been used to assess the base selectivity of an extensive series of these dyes. Circular dichroism measurements have shown that bis-intercalation is the predominant binding mode for the POPO-1 dye.

Working with Cyanine Dimers

All of the dyes in the TOTO series (Table 8.2) are supplied as 1 mM solutions in dimethylsulfoxide (DMSO), except for POPO-3 (P3584), which is supplied as a 1 mM solution in dimethylformamide (DMF). These cationic dyes appear to be readily adsorbed out of aqueous solutions onto surfaces (particularly glass) but are very stable once complexed to nucleic acids. Several applications of these dyes for staining nucleic acids in solutions, gels, microarrays and cells are described in Section 8.3, Section 8.5, Section 8.6, Section 8.7 and Section 15.5.

Cell-Impermeant Cyanine Monomers: The TO-PRO Family of Dyes

Our Patented TO-PRO family of dyes (Monomeric Cyanine Nucleic Acid Stains), all of which are listed in Table 8.2, each comprise a single cyanine dye and a cationic side chain (). The eleven dyes in the TO-PRO series are spectrally analogous to the corresponding dimeric cyanine dyes; however, with only two positive charges and one intercalating unit, the TO-PRO dyes exhibit somewhat reduced affinity for nucleic acids relative to the dyes in the TOTO series. Like their dimeric counterparts, these monomeric cyanine dyes are typically impermeant to cells, although the YO-PRO-1 (Y3603) dye has been shown to be permeant to apoptotic cells, providing a convenient indicator of apoptosis (Section 15.5, Figure 15.85). YO-PRO-1 has also been observed to pass through P2X₇ receptor channels of live cells.

Spectral Characteristics of the Cyanine Dye Monomers

The TO-PRO family of dyes retains all of the exceptional spectral properties of the dimeric cyanine dyes discussed above. The absorption and emission spectra of these monomeric cyanine dyes cover the visible and near-infrared spectrum (Table 8.2). They also have relatively narrow emission bandwidths, thus facilitating multicolor applications in imaging and flow cytometry. The YO-PRO-1 (491/509) and TO-PRO-1 (515/531) dyes are optimally excited by the 488 nm and 514 nm spectral lines of the argon-ion laser, respectively. In flow cytometric analysis, the TO-PRO-3 (642/661) complex with nucleic acids has been excited directly by the red He–Ne laser and indirectly by the argon-ion laser by using fluorescence resonance energy transfer (FRET) from co-bound propidium iodide (Technical Focus: Fluorescence Resonance Energy Transfer (FRET)). The TO-PRO-3 complex with nucleic acids has also been detected in a flow cytometer equipped with an inexpensive 3 mW visible-wavelength diode laser that provides excitation at 635 nm. Although the DNA-induced fluorescence enhancement of the TO-PRO-5 dye (T7596) is not as large as that observed with our other cyanine dyes, its spectral characteristics (excitation/emission maxima ~745/770 nm) provide a unique alternative for multicolor applications.

Working with Cyanine Monomers

The binding affinity of the TO-PRO series of dyes to dsDNA is lower than that of the TOTO series of dyes but is still very high, with dissociation constants in the micromolar range. TO-PRO dyes also bind to RNA and ssDNA, although typically with somewhat lower fluorescence quantum yields. Fluorescence polarization studies indicate that the TO-PRO-1 and PO-PRO-1 dyes bind by intercalation, with unwinding angles of 2° and 31°, respectively. Binding of these dyes to dsDNA is not sequence selective. All dyes of the TO-PRO series (Table 8.2) are supplied as 1 mM solutions in DMSO. Various applications of the TO-PRO series of dyes for staining nucleic acids are described in Section 8.3, Section 8.5, Section 8.6 and Section 15.5.

Cell-Impermeant SYTOX Dyes for Dead-Cell Staining

Our three SYTOX nucleic acid stains (Table 8.2) are cell-impermeant cyanine dyes that are particularly good dead-cell stains. These SYTOX stains are included in our RediPlate 96 nucleic acid stain sampler microplate (R32715), which is described below.

SYTOX Green Stain

The SYTOX Green nucleic acid stain (S7020, SYTOX(R) Green Nucleic Acid Stain) is a high-affinity nucleic acid stain that easily penetrates cells with compromised plasma membranes and yet will not cross the membranes of live cells. It is especially useful for staining both gram-positive and gram-negative bacteria — and probably virus particles — where an exceptionally bright signal is required. Following brief incubation with the SYTOX Green stain, dead cells fluoresce bright green when excited with the 488 nm spectral line of the argon-ion laser or with any other 450–500 nm source. No wash steps are required, since all of the SYTOX dyes are essentially nonfluorescent in aqueous medium. Unlike the DAPI or Hoechst dyes, the SYTOX Green nucleic acid stain shows little base selectivity. These properties, combined with its ~1000-fold fluorescence enhancement upon nucleic acid binding and high quantum yield, make our SYTOX Green stain a simple and quantitative single-step dead-cell indicator for use with epifluorescence and confocal laser-scanning microscopes, fluorometers, fluorescence microplate readers and flow cytometers (Figure 15.11). The SYTOX Green dye is included as a dead-cell stain in our Vybrant Apoptosis Assay Kits #1, #8, #9 and #10 (V13240, V35112, V35113, V35114; Section 15.5), in our ViaGram Red⁺ Bacterial Gram Stain and Viability Kit (V7023, Section 15.3) and in combination with C₁₂-resazurin in our LIVE/DEAD Cell Vitality Assay Kit (L34951, Section 15.3).

The SYTOX Green nucleic acid stain can be used with blue- and red-fluorescent labels for multiparameter analyses (). It is also possible to combine the SYTOX Green nucleic acid stain with the SYTO 17 red-fluorescent nucleic acid stain (S7579) for two-color visualization of dead and live cells (Section 15.3). Because the SYTOX Green nucleic acid stain is an excellent DNA counterstain for chromosome labeling and for fixed cells and tissues (), we have incorporated it into our Cytological Nuclear Counterstain Kit (C7590), which is discussed in Section 8.6.

SYTOX Blue Stain

Our SYTOX Blue stain (5 mM solution in dimethylsulfoxide (DMSO), S11348, SYTOX(R) Blue Nucleic Acid Stain; 1 mM solution in DMSO, S34857, SYTOX(R) Blue Dead Cell Stain) is a high-affinity nucleic acid stain that typically penetrates only cells with compromised plasma membranes (). The SYTOX Blue stain labels both DNA and RNA with extremely bright fluorescence centered near 470 nm (). The absorption maximum of the nucleic acid–bound SYTOX Blue stain (~445 nm) permits very efficient fluorescence excitation by the 436 nm spectral line of the mercury-arc lamp. Unlike many blue-fluorescent dyes, the SYTOX Blue stain is also efficiently excited by tungsten–halogen lamps and other sources that have relatively poor emission in the UV portion of the spectrum. The brightness of the SYTOX Blue stain allows sensitive detection with fluorometers, microplate readers, arc-lamp–equipped flow cytometers and epifluorescence microscopes, including those not equipped with UV-pass optics.

In a side-by-side comparison with the SYTOX Green stain, the SYTOX Blue stain yielded identical results when quantitating membrane-compromised bacterial cells. Furthermore, like the SYTOX Green stain, the SYTOX Blue stain does not interfere with bacterial cell growth. Because their emission spectra overlap somewhat, we have found that it is not ideal to use the SYTOX Blue stain and green-fluorescent dyes together; however, fluorescence emission of the SYTOX Blue stain permits clear discrimination from orange- or red-fluorescent probes, facilitating the development of multicolor assays with minimal spectral overlap between signals.

SYTOX Orange Stain

Our SYTOX Orange nucleic acid stain (S11368, SYTOX(R) Orange Nucleic Acid Stains) clearly distinguishes dead bacteria, yeast or mammalian cells. The SYTOX Orange stain has shorter-wavelength emission, as compared with propidium iodide, and its spectra more closely matches the rhodamine filter set (). In addition, the SYTOX Orange stain has a much higher molar absorptivity (extinction coefficient) than propidium iodide and a far greater fluorescence enhancement upon binding DNA, suggesting that it may have a higher sensitivity as a dead-cell stain or as a nuclear counterstain. The SYTOX Orange stain was shown to be the best dye for DNA fragment sizing by single-molecule flow cytometry when using a Nd:YAG excitation source, with a 450-fold enhancement upon binding to dsDNA.

Cell-Permeant Cyanine Dyes: The SYTO Nucleic Acid Stains

SYTO Nucleic Acid Stains for DNA and RNA

The numerous Patented SYTO dyes in Table 8.3 are somewhat lower-affinity nucleic acid stains that passively diffuse through the membranes of most cells. These UV- or visible light–excitable dyes can be used to stain RNA and DNA in both live and dead eukaryotic cells, as well as in gram-positive and gram-negative bacteria. Molecular Probes has synthesized a large number of SYTO dyes (Table 8.3) that share several important characteristics:

• Permeability to virtually all cell membranes, including mammalian cells and bacteria (Chapter 15)
• High molar absorptivity, with extinction coefficients greater than 50,000 cm^-1M^-1 at visible absorption maxima
• Extremely low intrinsic fluorescence, with quantum yields typically less than 0.01 when not bound to nucleic acids
• Quantum yields typically greater than 0.4 when bound to nucleic acids

Available as blue-, green-, orange- or red-fluorescent dyes, these novel SYTO stains provide researchers with visible light–excitable dyes for labeling DNA and RNA in live cells (). The SYTO dyes may also be useful for nucleic acid detection in solution, in electrophoretic gels, on blots, on microarrays and in several other assays. SYTO dyes differ from each other in one or more characteristics, including cell permeability, fluorescence enhancement upon binding nucleic acids, excitation and emission spectra (Table 8.3), DNA/RNA selectivity and binding affinity. The SYTO dyes are compatible with a variety of fluorescence-based instruments that use either laser excitation or a conventional broadband illumination source (e.g., mercury- and xenon-arc lamps).

The SYTO dyes can stain both DNA and RNA. In most cases, the fluorescence wavelengths and emission intensities are similar for solution measurements of DNA or RNA binding. Exceptions that we know of include the SYTO 12 and SYTO 14 dyes, which are about twice as fluorescent when complexed with RNA as with DNA, and SYTO 16, which is about twice as fluorescent on DNA than RNA. Consequently, the SYTO dyes do not act exclusively as nuclear stains in live cells and should not be equated in this regard with DNA-selective compounds such as DAPI or the Hoechst 33258 and Hoechst 33342 dyes, which readily stain cell nuclei at low concentrations in most cells. SYTO dye–stained eukaryotic cells will generally show diffuse cytoplasmic staining, as well as nuclear staining. The SYTO 14 dye (S7576) has been used to visualize the translocation of endogenous RNA found in polyribosome complexes in living cells. Particularly intense staining of intranuclear bodies is frequently observed. Because these dyes are generally cell permeant and most of the SYTO dyes contain a net positive charge at neutral pH, they may also stain mitochondria. In addition, the SYTO dyes will stain most gram-positive and gram-negative bacterial cells. Dead yeast cells are brightly stained with the SYTO dyes, and live yeast cells typically exhibit staining of both the mitochondria and the nucleus. Some of the SYTO dyes have been reported to be useful for detecting apoptosis (Section 15.5), and dyes structurally similar to the SYTO dyes have been used to detect multidrug-resistant cells (Section 15.6). The red-fluorescent SYTO dyes are proving useful as counterstains (Section 8.6) when combined with green-fluorescent antibodies (Section 7.2), lectins (Section 7.7) or the cell-impermeant SYTOX Green nucleic acid stain (see above). Several of the green-fluorescent SYTO dyes are excellent nuclear counterstains. We anticipate that many more applications will be found for these unique nucleic acid stains.

All of the Patented SYTO dyes are available separately (Table 8.3), and several SYTO dyes are included in our LIVE/DEAD Kits (Section 15.3, Table 15.2) and in our Bacteria Counting Kit (B7277, Section 15.4). The green-fluorescent SYBR 14 dye, a component of our LIVE/DEAD Sperm Viability Kit (L7011, Section 15.3) is also in the SYTO family of dyes. To facilitate testing the SYTO dyes in new applications, we offer several sampler kits containing sample sizes of SYTO dyes in each color set (Table 8.3), as well as the RediPlate 96 nucleic acid stain sampler microplate (R32715, described below), which includes 36 different SYTO dyes. With each purchase of a sampler kit or individual reagent we include a detailed product information sheet, describing the spectral properties of the dyes, to assist the researcher in designing staining protocols. The recommended dye concentration for cell staining depends on the assay and may vary widely but is typically 1–20 µM for bacteria, 1–100 µM for yeast and 10 nM–5 µM for other eukaryotes.

SYTO RNASelect Green-Fluorescent Cell Stain

SYTO RNASelect green-fluorescent cell stain (S32703, Section 15.2) is a cell-permeant nucleic acid stain that selectively stains RNA (Figure 15.18). Although virtually nonfluorescent in the absence of nucleic acids, the SYTO RNASelect stain exhibits bright green fluorescence when bound to RNA (absorption/emission maxima ~490/530 nm), but only a weak fluorescent signal when bound to DNA (Figure 8.12). Filter sets that are suitable for imaging cells labeled with fluorescein (FITC) will work well for imaging cells stained with SYTO RNASelect stain (, ).

Chemically Reactive Cyanine Dyes

The amine-reactive succinimidyl esters of the SYBR 101, SYBR 102 and SYBR 103 dyes (S21500, S21501, S21502) can be conjugated to peptides, proteins, drugs, polymeric matrices and biomolecules with primary amine groups. The conjugates are expected to be essentially nonfluorescent until they are able to complex with nucleic acids, resulting in strong green fluorescence. Thus, they may be useful for studies of nucleic acid binding to various biomolecules, such as DNA-binding proteins. It is also possible that the fluorescence enhancement upon nucleic acid binding of reactive SYBR dye conjugates will be useful for monitoring their transport into the nucleus. SYBR dye conjugates of solid or semisolid matrices (such as microspheres, magnetic particles or various resins) may be useful for detection or affinity isolation of nucleic acids.

The reactive SYBR dyes may also be conjugated to amine-modified nucleic acids. Although it is possible that the SYBR dyes may show some fluorescence when conjugated to amine groups on nucleic acids, they may be useful for developing homogeneous hybridization assays in which a specific sequence can be quantitated in solution without the need to separate bound and free probes. For example, a similar reactive nucleic acid stain has been used to label peptide–nucleic acid conjugates (PNA) for use as probes in real-time PCR. The labeled PNA probes exhibited a fluorescence increase upon hybridization to their complementary sequence and have been used to identify a single-base mismatch in a 10-base target sequence.

RediPlate 96 Nucleic Acid Stain Sampler Microplate

The SYTO dyes are relatively low-affinity nucleic acids stains that passively diffuse through the membranes of most cells. Like the structurally similar SYBR Gold, SYBR Green and SYBR Safe nucleic acid stains (Section 8.4), these UV- or visible light–excitable dyes can be used to stain RNA and DNA in both live and dead eukaryotic cells, as well as in gram-positive and gram-negative bacteria. The SYTO dyes may also be useful for nucleic acid detection in solution, in electrophoretic gels, on blots, on microarrays and in many other applications. Because of their relatively low nucleic acid–binding affinity, SYTO dyes stain a wider variety of cellular targets than do dyes such as Hoechst 33342, YO-PRO-1 and YOYO-1, and the cellular staining behavior of SYTO dyes can be variable and difficult to predict a priori. Consequently, extensive dye screening is beneficial in developing new applications for these dyes.

The RediPlate 96 nucleic acid stain sampler microplate (R32715) is designed to facilitate the screening of nucleic acid stains for new applications by providing samples of 36 different SYTO dyes predispensed in a 96-well microplate. The plate also contains samples of the SYBR Green I, SYBR Green II and PicoGreen dyes. Although these latter three dyes were primarily developed for detecting nucleic acids in electrophoretic gels or in solution, they have also proven useful in cellular staining applications. Also included are samples of the amine-reactive SYBR 101 and SYBR 103 nucleic acid–binding dyes. Finally, samples of six other nucleic acid–binding dyes (Hoechst 33342, SYTOX Green, SYTOX Orange, SYTOX Blue, propidium iodide and hexidium iodide) are provided for indicating cell viability and as references for the cellular staining behavior of the SYTO dyes.

Each RediPlate 96 nucleic acid stain sampler microplate consists of one 96-well microplate containing duplicate samples of 47 different nucleic acid–binding dyes and two empty wells for fluorescence background measurements. The amount of dye in each well is calibrated to yield a concentration of 20 µM after solubilization in 100 µL of a suitable solvent, typically dimethylsulfoxide (DMSO) or aqueous buffer. The general characteristics of the dyes provided in the RediPlate 96 nucleic acid stain sampler microplate are summarized in Table 8.5 and described in detail in the accompanying product information sheet (RediPlate 96 Nucleic Acid Stain Sampler Microplate).

Phenanthridines and Acridines: Classic Intercalating Dyes

Cell-Impermeant Ethidium Bromide and Propidium Iodide

Ethidium bromide (EtBr, E1305; E3565; ) and propidium iodide (PI, P1304MP; P3566; FluoroPure Grade, P21493; ; Propidium Iodide Nucleic Acid Stain) are structurally similar phenanthridinium intercalators. PI is more soluble in water and less membrane-permeant than EtBr, although both dyes are generally excluded from viable cells. EtBr and PI can be excited with mercury- or xenon-arc lamps or with the argon-ion laser, making them suitable for fluorescence microscopy, confocal laser-scanning microscopy (), flow cytometry and fluorometry. These dyes bind with little or no sequence preference at a stoichiometry of one dye per 4–5 base pairs of DNA. Excitation of the EtBr–DNA complex may result in photobleaching of the dye and single-strand breaks. Both EtBr and PI also bind to RNA, necessitating treatment with nucleases to distinguish between RNA and DNA. Once these dyes are bound to nucleic acids, their fluorescence is enhanced ~10-fold, their excitation maxima are shifted ~30–40 nm to the red and their emission maxima are shifted ~15 nm to the blue (Figure 8.16, Table 8.4). Although their molar absorptivities (extinction coefficients) are relatively low, EtBr and PI exhibit sufficiently large Stokes shifts to allow simultaneous detection of nuclear DNA and fluorescein-labeled antibodies, provided that the proper optical filters are used