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	WARNING: The information provided in these pages is copyrighted and is intended for individual use only. No parts of this work (text, tables or pictures) may be commercialized, published or otherwise reproduced without the written consent of the author. Ref: Nature Biotechnology 2000, Vol 18, p345-348. Get article in PDF format here

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1. Chemical coupling protocol(s)

Introductory note: the custom fluorescent nucleotide synthesis, published in Nature Biotech 18:345-348 (2000) and described in more detail here, is in constant development, and the protocols are subject to improvement. As simple as the procedure is, it is likely that through the contribution of the people who use it, the efficiency of both the chemical coupling and nucleic acid labeling protocols will be further improved. If you have suggestions for any of the protocols described, and would like other people to take advantage of your knowledge, please feel free to let me know and I will add your suggestions to the protocol, while giving you full credit for your work or ideas.
That the protocols need further improvement is quite obvious, as there are several dye-dUTP combinations which I could not use for DNA labeling (AMCA, Cascade Blue, Cy3.5, Cy5.5). However, companies such as Molecular Probes or Amersham-Pharmacia sell dUTP or dCTP conjugated to these dyes, meaning that these labeled nucleotides can be used. Whether this means adding a spacer between the dye and the nucleotide or, maybe, changing simpler things such as the pH of a reaction, any suggestions backed by some experimental proof are welcome. Replacing BSA with a chemical compound (probably an anti-oxidant of some kind) which does not inhibit DNA labeling reactions and does not require removal prior to DNA precipitation would be beneficial.

1.1. Generalities

Generally, labeled nucleotides are synthesized by chemically coupling allylamine-dUTP to succinimidyl-ester derivatives of fluorescent dyes (see table below) or haptenes (Biotin, Digoxigenin, Dinitrophenyl - these require fluorescently-labeled antibodies or specific proteins for visualization/detection). Various companies also include a spacer (usually 5-16 carbon atoms long) between the dye and the nucleotide, which seems to improve enzymatic incorporation of the modified nucleotides. The custom-made labeled nucleotides presented here usually do not have a spacer, unless one is provided by the company synthesizing the reactive dye/haptene (the spacer is denoted by the symbol "X" - see the bottom of this page).
The following fluorescent dyes were tested: amino-methyl coumarin (AMCA); diethyl aminomethyl coumarin (DEAC); Cascade Blue (CB); fluorescein isothiocyanate (FITC); Oregon Green (OG); Alexa 488 (A488); Rhodamine green (RGr); Carboxy-rhodamine 6G (R6G); tetramethyl rhodamine (TAMRA); Texas Red (TxR); Cy3; Cy3.5; Cy5, Cy5.5 and carboxynaphtofluorescein (CNF). Cy7 was used only for protein labeling. The haptenes tested were biotin (BIO); digoxigenin (DIG); and 2,4-dinitrophenyl (DNP). RGr and CNF were tested after the publication was in press.

The succinimidyl ester derivatives of the dyes and haptenes, as well as the reactive nucleotide (allylamine-dUTP), are all available commercially as dry powders, in 5-10-25 mg aliquots. Most of the dyes can be dissolved in DMSO, but some (the acetylazide derivative of Cascade Blue and the succinimidyl-ester derivative of Alexa 488) are soluble in water.
The volume of solvent (DMSO, water) in microliters (ul) required to dissolve the dyes, haptenes or allylamine dUTP at the desired molarity, can be calculated according to the following formula:

Reactive dyes were dissolved at the concentration shown in the table below (between 10-40 mM). All dyes were dissolved in DMSO, with the exception of CB and A-488.
Commercially available allylamine-dUTP [5-(3-aminoallyl)-2'-deoxyuridine 5' triphosphate (Sigma, St. Louis, MO)] was dissolved in 0.2 M bicarbonate buffer (~pH 8.3) at 20mM concentration.

1.2. Chemical coupling protocols

Chemical coupling reactions were carried out at 1:1 molar concentration (dye : dUTP) in 80-100 mM bicarbonate buffer, adding the allylamine-dUTP (20 mM stock solution, in 0.2M bicarbonate buffer) first, and the reactive dye last.

Table 1. Dyes and coupling protocols.
Note: your web browser may not allow you to visualize or print the color version of the table. Click here to see Table 1 in JPG format (JPG files can be printed in colors or downloaded to your computer).

Table 1 legend: No 1-8 in the first column indicate the eight groups of fluors across the visible and infrared spectrum, which can be detected using a fluorescence microscope equipped with the corresponding 8 fluorescence filters (Chroma Technologies). The colors in the table indicate with approximation the color of the respective fluorescent dyes (Cy5,Cy5.5 and Cy7 have absorption and emission peaks in the infrared, and cannot be visualized directly. Thus, their assigned color in the table is arbitrary). H1, H2 and H3 are the three haptenes (biotin, digoxigenin and dinitrophenyl) used. MW is the approximate molecular weight of the dyes. Abs = absorption and Em = emission peaks (NM) for the respective dyes. Dye (mM) indicates the concentration at which the reactive dye was dissolved in DMSO (or water) for use and long-term storage. The chemical coupling protocol requires mixing of four-five ingredients (numbers 1 through 5) in the sequence described in the table (the dye is always added last)."n" = any volume. "~dUTP" is the chemically active, allylamine-dUTP, dissolved at 20mM in 0.2M bicarbonate buffer. * or **: the DNP-ester precipitates easier than other dyes. Therefore, it is recommended that, using small amounts (1volume = 10uL), both protocols described in the table are tried. In our hands, one batch of dye worked well using the first protocol (*), whereas a second batch of dye worked when using the second protocol (**), in which the order in which the ingredients were added was changed (1-5). Please note that after mixing DNP (1), DMSO (2) and ~dUTP (3), the solution becomes "cloudy", but it clarifies after adding water (4) and 0.2M bicarbonate (5).

After 3-4 hours incubation at room temperature, to every reaction add in order:

- 0.2n 2M glycine (pH 8.0, 20 mM final concentration), to stop the reaction;
- 0.4n 1M Tris-HCl, pH 7.75 (20 mM final concentration), to stabilize the nucleotides;
- water to 20n, which brings the final dUTP concentration to 1mM.
A convenient labeling protocol uses 1n = 10 uL, and yields 200 uL labeled nucleotide solution (1 mM).

Previous data indicated that roughly 50% of the dUTP was labeled in such reactions. Nucleotides could be used immediately or stored at Ý20 C. Reactions carried out at a 2:1 molar excess of dye achieved higher dUTP labeling (80-90%) and are more cost-effective for the cheaper fluorescein, rhodamine and coumarine derivatives. Because of the higher costs of the cyanine dyes and to maintain uniformity, we carried out the reactions at 1:1 molar ratios. Solutions were stable for at least 1 year when stored at Ý20 C.

1.3. Source of various reagents (* = chemically active).

The color indicates the corresponding abbreviated name in Table 1 above.
NOTE: purchasing bulk quantities of cyanine dyes from Amersham/Pharmacia requires signing of a formal agreement with the company. Please contact your local representative or the company for details.

Other dyes tested:

AMCA (Molecular Probes, Cat# 6118)
Cascade Blue (CB) (Molecular Probes, Cat# C-2284)
Oregon Green 488 (OG) (Molecular Probes, Cat# O-6147 or O 6149)
Alexa Fluor 488 (A-488) (Molecular Probes, Cat# A-10235 = protein labeling kit)
Carboxynaphtofluorescein (CNF) (Molecular Probes, Cat# C-653)

The following dyes either did not work well for nucleotide labeling or, more likely, although chemical coupling probably worked, the corresponding labeled dUTP could not be incorporated by enzymes: AMCA; Cascade Blue; Cy3.5; Cy5.5; Oregon Green; CNF. Cy7 was not tested for nucleotide labeling. All these dyes, however, worked when conjugated to antibodies or avidin.