Nucleic Acid Staining: Definition, Principles, Dyes, Procedures, and Uses

Nucleic acid dyes are compounds that can bind to nucleic acids and are commonly used for the quantification and detection of nucleic acids. These dyes are generally aromatic compounds containing benzene rings or pyridine rings. These ring structures absorb ultraviolet or blue light and emit fluorescence. Nucleic acid molecules are composed of nucleotide units, which consist of a five-carbon sugar, a nitrogenous base, and a phosphate group. DNA and RNA differ, but both contain four types of bases. Nucleic acid dyes typically stain by interacting with one or more of these bases.

Nucleic Acid Definition

Nucleic acids are among the most important biomolecules in living organisms, serving as the material basis for the storage, transmission, and expression of genetic information. They include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA is usually found in the cell nucleus, with small amounts present in mitochondria or chloroplasts. Its functions include storing genetic information, regulating genetic expression, transcription of genetic information, and subsequent expression. RNA is mainly found in the cytoplasm and directly performs the conversion of genetic information into proteins. For example, mRNA expresses genetic information from DNA into proteins, tRNA transports specific amino acids for protein synthesis, and rRNA participates in catalysis within ribosomes. In viruses, RNA can serve as genetic material.

Fig. 1. Structure of nucleic acid.

Nucleic Acid Structure

From a spatial structural perspective, DNA single strands are formed by the polymerization of four deoxyribonucleotides (A, G, C, and T) in a specific sequence through a phosphodiester backbone, forming the primary structure of DNA. RNA single strands, formed by the polymerization of four ribonucleotides (A, G, C, and U) in a specific sequence, form the primary structure of RNA. However, the key difference between DNA and RNA lies not only in their primary structural chemical composition but also in their secondary structures. RNA typically exists as a single strand, while DNA forms a double helix through the complementary pairing of two reverse DNA strands, exhibiting the characteristic double helix structure.

How Do Nucleic Acid Stains Work?

Fluorescence detection technology offers advantages such as speed, non-destructiveness, and real-time detection, making it widely used in nucleic acid testing. The binding of nucleic acid dyes to nucleic acids is primarily accomplished through electrostatic interactions, hydrogen bonding, and hydrophobic interactions. Nucleic acids typically carry a negative charge, while nucleic acid dyes generally carry a positive charge, so they bind through electrostatic attraction. Some groups on the nucleic acid dyes can also form hydrogen bonds with the bases of nucleic acids, thus binding with the nucleic acids. Additionally, nucleic acid dyes are generally insoluble in water, and hydrophobic dyes can interact with hydrophobic bases in nucleic acids. When they bind, the fluorescence properties of the dye change, and this change forms the basis for the quantification or qualitative analysis of nucleic acids.

Nucleic Acid Dyes

Nucleic acid stains are typically aromatic compounds containing benzene or pyridine rings in their molecular structure. These ring structures can absorb ultraviolet or blue light and emit fluorescence. Nucleic acid dyes are generally positively charged, while nucleic acids (DNA and RNA) are negatively charged, allowing for electrostatic attraction between the two. Based on staining characteristics, nucleic acid dyes can be classified into types such as non-permeable, permeable, and amino-reactive dyes. Based on the structural characteristics of the dye, nucleic acid dyes can also be categorized into cationic dyes, basic dyes, and other types.

• Ethidium Bromide (EB)

Ethidium Bromide (EB) is one of the earliest and most commonly used nucleic acid dyes. It can intercalate between the base pairs of the DNA double helix, forming a stable EB-DNA complex. Under ultraviolet light, this complex emits an orange-red fluorescence, allowing nucleic acid fragments to be clearly visible after electrophoresis. Its sensitivity is sufficient for most laboratory nucleic acid detection needs. Ethidium Bromide is relatively inexpensive, which makes it widely used in laboratories, particularly in those with limited budgets, where it is often the first choice. However, it should be noted that EB is highly carcinogenic and mutagenic, posing significant potential harm to humans. Therefore, strict safety measures such as wearing gloves and masks must be followed, and waste must be properly disposed of after use.

• Goldview Series Dyes

Goldview is another novel nucleic acid dye that can replace Ethidium Bromide. When used for DNA detection in agarose gel electrophoresis, it binds to DNA and produces strong fluorescence signals, with sensitivity comparable to EB. Under UV transmission light, double-stranded DNA (dsDNA) emits green fluorescence, while single-stranded DNA (ssDNA) emits red fluorescence. Although Goldview has lower toxicity than EB, it still exhibits some cytotoxicity, so protective measures are required during use. Goldview is particularly suitable for detecting larger DNA fragments (>1 kb), with high sensitivity, and can also be used for RNA staining. However, its fluorescence quenching under UV light is a limitation, and gels containing Goldview are not suitable for gel recovery experiments.

• SYBR Series Dyes

The SYBR series fluorescent dyes were introduced by Molecular Probes in 1995 as high-sensitivity nucleic acid stains. These dyes are asymmetrical cyanine compounds with high affinity for nucleic acids. The SYBR series includes SYBR Green I, SYBR Green II, and SYBR Gold dyes.

• SYBR Green I specifically binds to the minor groove of all dsDNA double helices and has a green excitation wavelength. In its free form, SYBR Green I emits weak fluorescence, but when bound to double-stranded DNA, fluorescence is significantly enhanced. Therefore, the fluorescence signal strength of SYBR Green I is correlated with the amount of double-stranded DNA, enabling the detection of PCR products. SYBR Green I has an absorption maximum around 497 nm and an emission maximum around 520 nm, making it one of the first alternatives to EB in nucleic acid detection.
• SYBR Green II is primarily used for staining RNA or single-stranded DNA. It binds to single-stranded nucleic acids with twice the efficiency of double-stranded DNA, allowing detection of extremely low nucleic acid concentrations and providing higher sensitivity than traditional dyes such as EB. It does not require decolorization or washing steps, simplifying the experimental process.
• SYBR Gold is a highly sensitive, low mutagenic nucleic acid gel stain. Its mutagenicity is much lower than that of EB, but its sensitivity is 25-100 times higher than EB, able to detect as low as 25 pg of DNA. When bound to nucleic acids, the signal is enhanced by over 1000 times, making detection more accurate. SYBR Gold can detect dsDNA, ssDNA, and RNA in non-denaturing, ethylene glycol, formaldehyde, or urea gels and has a wide range of applications. Compared to EB, SYBR Gold has a lower mutagenic effect and is safer for both humans and the environment.

• GelRed Series Dyes

GelRed is a sensitive, stable, and relatively safe fluorescent dyes. GelRed dyes mainly refer to GelRed and GelGreen, which can replace the highly toxic dye Ethidium Bromide (EB) for staining dsDNA and ssDNA in agarose or polyacrylamide gels. GelRed has much higher sensitivity than EB and does not require decolorization. Since GelRed shares the same spectral properties as EB, there is no need to replace imaging systems when substituting EB with GelRed. GelRed can be used for pre-made gels or post-electrophoresis staining. Typically, post-electrophoresis staining is more sensitive and eliminates interference with DNA migration. Using GelRed for post-electrophoresis staining is simple, requires no decolorization or special solutions. After diluting the dye in 0.1M NaCl and immersing the gel in the staining solution for 30 minutes, the results can be observed. The staining solution is highly stable at room temperature and can be reused multiple times. In comparison, pre-made gels require less dye, making the process simpler and more economical.

• EvaGreen

EvaGreen is a green fluorescent nucleic acid dye that exhibits lower PCR inhibition compared to SYBR Green due to its intelligent "on-demand release" DNA binding technology. This means that EvaGreen interferes less with DNA amplification in PCR reactions, thereby improving the sensitivity and accuracy of the experiment. The low PCR inhibition allows for higher dye concentrations, resulting in better qPCR and melting curve analysis signals. This makes EvaGreen highly sensitive when detecting low-concentration DNA samples. EvaGreen has exceptional thermal stability, chemical stability, and photostability. In PCR buffers, EvaGreen remains stable at 95-100 °C for 48 hours without detectable dye degradation, and it is highly stable under both acidic and alkaline conditions. It also withstands repeated freeze-thaw cycles. EvaGreen is widely used in quantitative real-time PCR (qPCR), DNA melting curve analysis, thermophilic helicase-dependent amplification (tHDA) real-time monitoring capillary gel electrophoresis, high-resolution melting curve analysis (HRM), loop-mediated isothermal amplification (LAMP), and digital PCR.

• LC Green PLUS

LC Green PLUS is a saturated fluorescent dye specifically designed for high-resolution melting curve analysis, with significant applications in DNA sequence variation detection. The dye is added to samples before PCR reactions and binds to the DNA double helix, enabling detection of single base variations in the DNA through melting curve analysis. The dye has extremely high fluorescence intensity and can be used with other fluorescence-based PCR detection systems, such as the Roche LightCycler. Its excitation wavelength is 440-470 nm, and its emission wavelength is 470-520 nm, making it ideal for LightScanner melting curve analysis. LC Green PLUS is highly stable and maintains stable binding to DNA at high temperatures, making it particularly suitable for high-GC-content PCR products. It has minimal inhibitory effects on PCR reactions, making it a saturating dye that can detect multiple PCR products in a mixture during melting analysis.

Nucleic Acid Staining Procedure

The nucleic acid staining procedure generally includes the following steps. First, prepare the samples to be stained and fix the cell or tissue samples using an appropriate fixative (such as formaldehyde or ethanol) to ensure the stability of the nucleic acids. Next, mix the samples with the dye solution, commonly used nucleic acid dyes such as DAPI, Hoechst, or Ethidium Bromide, which can specifically bind to DNA or RNA and emit fluorescence. The staining reaction is usually performed at room temperature for 15-30 minutes, allowing the dye to pass through the cell membrane and bind to the nucleic acids. After staining, observe the samples under a fluorescence microscope to detect the distribution and morphology of the nucleic acids. To obtain clear images, it may be necessary to apply anti-fluorescence quenching agents during mounting.

• DNA Staining

DNA staining is primarily used to visualize DNA in cell nuclei or genomic DNA in electrophoresis samples. Commonly used dyes include DAPI, Hoechst, and Ethidium Bromide (EtBr), which generate fluorescence signals by intercalating between the base pairs of the DNA double helix or binding to the DNA grooves. Cell samples need to be fixed and permeabilized to allow the dye to enter the cell nucleus. For gel electrophoresis, EtBr or SYBR Green can be used for pre-staining or post-staining to label DNA bands, which are visible under UV light. DNA staining is simple, with stable signals, making it suitable for cell nucleus imaging and DNA quantification analysis.

• RNA Staining

RNA staining is specifically used for detecting and analyzing the distribution and content of RNA in samples. RNA dyes such as SYTO RNA-Select, Acridine Orange, and SYBR Green II specifically bind to single-stranded RNA molecules and emit fluorescence. During sample handling, it is important to avoid RNA degradation by RNase and maintain RNA integrity. When staining cells, the fixation and permeabilization steps must be performed gently to prevent RNA damage. In gel electrophoresis, RNA dyes are used to label RNA bands, which aids in assessing RNA integrity and quantification.

What is Nucleic Acid Staining Used For?

Nucleic acid staining is an important technique in molecular biology and cell biology research, widely applied for the detection, quantification, and localization of DNA and RNA. These dyes can specifically bind to nucleic acids, and with the help of equipment such as fluorescence microscopes or flow cytometers, they assist scientists in observing and analyzing the distribution, concentration, and changes of nucleic acids within cells. The following are some of the major application areas of nucleic acid staining:

• Cell Nucleus Observation and Morphological Analysis

Nucleic acid staining is extensively used in cytological research, particularly for observing cell nuclei. By using dyes such as DAPI (4',6-diamidino-2-phenylindole), researchers can clearly observe the morphology, number, and distribution of the cell nucleus. DAPI staining is commonly used in studies of the cell cycle, apoptosis, and nuclear changes during cell division. Due to DAPI's high affinity for DNA and its ability to emit blue fluorescence, it has become an important tool for detecting cell nuclei.

• DNA Localization and Chromosome Analysis

Nucleic acid staining is widely applied in chromosome analysis and nucleic acid localization. For example, dyes such as Hoechst 33342 or PI (Propidium Iodide) can clearly display the distribution and structure of chromosomes in cells. After staining, cells can be observed using fluorescence microscopy or flow cytometry, helping researchers analyze the number and morphology of chromosomes in the cell nucleus. This technique is extensively used in genomics and cytogenetics.

• RNA Detection and Analysis

RNA staining also plays an important role in nucleic acid staining. Specific dyes, such as SYTO RNA dyes, can be used to label RNA molecules in cells, which is crucial for studying gene expression and RNA localization. Through nucleic acid staining, researchers can observe the position and abundance of RNA molecules within cells, allowing them to infer gene transcription activity. For instance, combining nucleic acid staining with fluorescence in situ hybridization (FISH) enables precise localization of specific RNA molecules, revealing their spatial and temporal dynamics within cells.

• Cell Cycle and Cell Proliferation Studies

Nucleic acid staining is commonly used in cell cycle studies. After staining DNA with dyes, researchers can analyze the distribution of cells at different stages of the cell cycle using flow cytometry. For example, PI and Hoechst dyes are widely used in cell cycle analysis to distinguish between cells in different stages (G0/G1, S, G2, and M phases) based on fluorescence signal intensity. Additionally, nucleic acid staining is applied in cell proliferation studies, where the fluorescence intensity of the dye can be used to measure DNA synthesis, thus evaluating the rate of cell proliferation.

• Apoptosis Detection

Nucleic acid staining is a common technique in apoptosis research. During apoptosis, cells undergo DNA degradation, and dyes such as TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) and Hoechst can label these DNA breaks, helping to identify apoptotic cells. When combined with fluorescence microscopy, nucleic acid staining enables real-time observation of the apoptosis process and its changes under different experimental conditions.

• Cytoplasm and Nucleus Separation

Nucleic acid staining also plays an important role in the separation of the cytoplasm and nucleus. By using specific dyes, researchers can clearly mark the boundary between the cytoplasm and the nucleus, allowing for precise distinction between different parts of the cell. This is significant for studying the functions and interactions of various regions within the cell.

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