Fluorescent Dyes

Characteristics of 5 kinds of fluorescent dyes commonly used in nucleic acid detection

Nucleic acid fluorescent dyes stain the nucleus and quantitatively measure the fluorescence intensity of the cell, so that the content of DNA and RNA in the nucleus can be determined, and the cell cycle and cell proliferation can be analyzed. There are a variety of fluorescent dyes that can stain DNA or RNA in cells. Commonly used DNA dyes include propidium iodide (PI), DAPI, Hoechst 33342, etc., and RNA dyes include thiazole orange, acridine orange, etc.

1. Fluorescent dye PI (propidium iodide)

The fluorescent dye PI (propidium iodide) is a nuclear staining reagent that can stain DNA. It is often used for apoptosis detection. The full English name is Propidium Iodide. It is an ethidium bromide analog that emits red fluorescence upon intercalation in double-stranded DNA. Although PI cannot pass through living cell membranes, it can pass through damaged cell membranes and stain nuclei. PI is often used with fluorescent probes such as Calcein-AM or FDA to simultaneously stain live and dead cells. The excitation and emission wavelengths of the PI-DNA complex were 535 nm and 615 nm, respectively.

2. EB (ethidium bromide)

Ethidium bromide is a highly sensitive intercalating fluorescent dye, a strong carcinogenic mutagen, but it is cheap. Its binding to DNA has little base sequence specificity. In saturated solutions of high ionic strength, approximately one molecule of ethidium bromide is inserted every 2.5 bases. This property of EB makes it a nucleic acid dye commonly used for nucleic acid staining in agarose gel electrophoresis. Ethidium bromide has absorption peaks at 302nm and 366nm in the ultraviolet region. Under the irradiation of ultraviolet light, ethidium bromide can be excited to orange-red fluorescence (590nm). Ethidium bromide complexes with DNA bound fluoresce 20-30 times more intensely than dyes that do not bind DNA, so ethidium bromide can detect DNA bands as small as 10 ng and is very sensitive.

3. Gel Green and Gel Red

The EB replacement dye developed and launched by Biotium has similar dyeing effect as EB, and is not easy to be quenched under UV light, so it can be used for glue recovery. But the price is more expensive.

GelRed and GelGreen are two excellent fluorescent nucleic acid gel staining reagents that combine high sensitivity, low toxicity, and ultra-stability. Its water-soluble dyes have passed the US EPA safety certification, and the waste can be directly poured into the sewer without causing any environmental pollution.

Its features are:
1) High sensitivity: GelRed and GelGreen are one of the most sensitive gel nucleic acid dyes in the market;
2) Excellent stability: it can be heated in a microwave oven and can be stored at room temperature;
3) Safer: Ames’ test results show that the mutagenicity of the dye is far less than that of ethidium bromide (EB);
4) Wide range of adaptability: suitable for precast gels and post-gel electrophoresis staining;
5) Simple staining process: like EB, there is no need to worry about dye degradation during precast gel and electrophoresis; and the staining process after electrophoresis only takes 30 minutes without destaining or washing;
6) Small impact on DNA and RNA migration: less impact on nucleic acid migration than SYBR Green I?;
7) Perfectly compatible with standard gel imaging systems and gel observation devices excited by visible light: GelRed can perfectly replace EB when using a UV gel imaging system excited by 312nm; GelGreen is sufficient to replace either SYB dye in gel viewing devices. However, this dye makes small DNA fragments migrate slower (compared to EB).

4. Gold View I/II

Goldview has a good staining effect for large DNA fragments, but it is not very good for fragments below 500bp. Another fatal weakness is that its fluorophore is very easy to quench under UV light, usually 5-10min. will disappear. Also Goldview is not suitable for glue recycling. Goldview is actually AO (acridine orange, a highly toxic product), and its fluorescence effect is far inferior to that of EB, with poor sensitivity, serious background color, unsuitable for recycling, unstable, and its ability to induce mutation under ultraviolet light is extremely high.

5. SYBR Green and SYBR Gold

Poor stability and toxicity. It belongs to anthocyanidin nucleic acid dyes. anthocyanidin dyes are not non-toxic, but only low-toxic. Electrophoresis grades are modified. There are three kinds of modifications: 1. Add halogen or cyano group. 2. Insertion of cyclic hydroxyl groups. 3. Add stabilizer. SYBR Green I is a highly sensitive DNA fluorescent dye suitable for a variety of electrophoretic analyses with simple operation: no destaining or washing required. At least 20pg DNA can be detected, which is 25-100 times higher than that of EB staining. When SYBR Green I binds to dsDNA, the fluorescence signal will be enhanced by 800-1000 times. The gel samples stained with SYBR Green I have strong fluorescence signal and low background signal. SYBR Green and SYBR Gold are much less stable than EB. The SYBR series of dyes can also enter cells to stain mitochondria and DNA in the nucleus, causing harm. SYBR Green I has been shown to have strong mutagenic ability under UV light, mutating DNA or other substances.

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