Nucleic Acid Labeling Support—Getting Started

Please see the table below for a comparison of our nucleic acid labeling methods.

The kits have been optimized for labeling 50 μg of a 5’-amine-modified oligonucleotide that is 18 to 24 bases in length per reaction. Slightly shorter or longer oligonucleotides may be labeled by the same procedure; however, you may need to adjustment the protocol for greatly shorter or longer oligonucleotides. We have not tested the kits with oligonucleotides containing more than one amine.

The kits include our Alexa Fluor® amine-reactive succinimidyl or tetrafluorophenyl (TFP) ester dyes, which can be used for covalent conjugation to a primary amine.

The exact amount of reactive dye per vial is proprietary; however, each vial provides an optimal amount of dye for labeling 50 μg of a 5’-amine–modified oligonucleotide that is 18 to 24 bases in length. The kit includes 3 vials of dye for 3 reactions total. The amine-reactive Alexa Fluor® dyes can also be purchased as stand-alone reagents in amounts ranging from 100 µg to 25 mg.

We recommend gel electrophoresis or reverse-phase HPLC after an ethanol precipitation step. More details on the purification procedure can be found in the product manual:

Alexa Fluor® Oligonucleotide Amine Labeling Kits Product Manual

• An amine-modified oligonucleotide can be obtained from commercial suppliers of custom oligonucleotides. For information about our custom DNA oligo service, click here.

ChromaTide® dye-labeled dUTP and UTP nucleotides can be used to synthesize labeled nucleic acid probes without the need for hazardous and expensive radioisotope-labeled nucleotides. You can incorporate these nucleotides using standard molecular biology techniques, and then use the labeled probes for in situ hybridization, microarrays, or blotting. ChromaTide® dye-labeled nucleotides are available with different fluorescent colors to facilitate multicolor analysis.

The ChromaTide® dUTP and UTP nucleotides are modified at the C-5 position of uridine via a unique alkynylamino linker, which provides a spacer between the nucleotide and the dye to reduce interactions between them. Several of these nucleotides also contain an additional spacer, further separating the label from the nucleic acid. The number in the product name, e.g., the “12” in fluorescein-12-dUTP, indicates the net length of the spacer, in atoms.

ChromaTide® dUTPs are most efficiently incorporated into DNA using nick translation, random primer labeling, end-labeling with terminal deoxynucleotidyl transferase (TdT), and reverse transcription. ChromaTide® UTPs can be incorporated into RNA using in vitro transcription. You can find example protocols using these enzymatic reactions below. Specific fluorescent ChromaTide® nucleotides may not be appropriate for certain enzymatic methods, so we recommend consulting Table 1 in the ChromaTide® product manual prior to purchase. Some ChromaTide® products have been discontinued, so not all of the listed ChromaTide® nucleotides may be currently available:

ChromaTide® Labeled Nucleotide Product Manual
Methods for Enzymatic Incorporation of ChromaTide® dUTPs
Methods for Enzymatic Incorporation of ChromaTide® UTPs

Although we have a protocol for incorporation using PCR, we do not recommend PCR as the best method for incorporation of any of our fluorescent or modified nucleotides. With PCR, incorporation rates are very low. We had some success with Taq polymerases in the past; however, many of the modern DNA polymerases are higher fidelity and make fewer mistakes; as a result, they do not incorporate modified nucleotides very well. The paper below provides a comparison of commercially available polymerases and PCR incorporation efficiency related to type of dye or modification:

Anderson JP, Angerer B, Loeb LA (2005) Incorporation of reporter-labeled nucleotides by DNA polymerases. Biotechniques 38:257–264.

The average incorporation is one dye for every 100–150 bases, so the ChromaTide® fluorescently labeled nucleotides typically produce the lowest labeling rates of the nucleic acid labeling methods we offer.

The base-to-dye ratio is determined by measuring the absorbance of the nucleic acid at 260 nm and the absorbance of the dye at its absorbance maximum. Using the extinction coefficients for the appropriate dye and nucleic acid, you can then calculate the base-to-dye ratio for the labeled nucleic acid using the Beer-Lambert law. Detailed instructions can be found in the product manuals below:

Methods for Enzymatic Incorporation of ChromaTide® dUTPs
Methods for Enzymatic Incorporation of ChromaTide® UTPs

No, they are not cell permeant so they are only suitable for in vitro incorporation methods. The fluorescent dyes and phosphate groups are too highly charged to allow the nucleotides to penetrate the membrane of an intact cell. Nonfluorescent nucleosides without phosphates such as EdU, EU, or BrdU can be used for live cell nucleic acid incorporation studies.

The ARES™ Alexa Fluor® DNA Labeling Kits provides a versatile, two-step method for labeling DNA with our Alexa Fluor® dyes. In the first step, an amine-modified nucleotide is incorporated into DNA using enzymatic labeling methods. In the second step, the amine-modified DNA is chemically labeled using our amine-reactive Alexa Fluor® dyes. The labeled probes can be used for:

• Fluorescence in situ hybridization (FISH). See Buster DW, Daniel SG, Nguyen HQ, et al. (2013) SCFSlimb ubiquitin ligase suppresses condensin II–mediated nuclear reorganization by degrading Cap-H2, J Cell Biol 201(1):49–63.
• Microarray techniques. See Shin HH, Hwang BH, Seo JH et al. (2014) Specific discrimination of three pathogenic Salmonella enterica subsp. enterica serotypes by carB-based oligonucleotide microarray. Appl Environ Microbiol 80(1):366–373.

Please see the image below for how the ARES™ Alexa Fluor® DNA Labeling Kits work.

A dye-to-base ratio of 1:12 to 1:35 is a suitable range for Alexa Fluor® dye labeling of cDNA for microarray and FISH (fluorescence in situ hybridization) applications.

At the same dye-to-base ratio, Alexa Fluor® dyes exhibit higher intensity and reduced self-quenching at higher labeling ratios. The image below compares the dye-to-base ratio and fluorescence of Alexa Fluor® 555 to Cy3, and Alexa Fluor® 647 to Cy®5 dye.

The following image compares the fluorescence emission and wavelength for the respective dyes.

Long-term storage for the ARES™-labeled probes can be done in just about any kind of buffer, TE, formamide, hybridization buffer, or ethanol. We suggest using your normal storage conditions as long as you protect the probes from light. ARES™ conjugates are very stable.

An ARES™-labeled oligonucleotide should survive at 95°C for 5 minutes.

Unfortunately, Alexa Fluor® 633 does not label nucleic acids well because of the dye's chemical structure. Furthermore, DNA probes labeled with Alexa Fluor® 633 do not form stable hybrids in nucleic acid hybridization assays.

Different preparations of RNA will certainly give different results. Most of the time, the mRNA is significantly degraded. The enzymatic incorporation of aminoallyl-dUTP (AA-dUTP) should not differ from reaction to reaction. If there are differences, it has to be due to the RNA or the method. AA-dUTP incorporation is no different than that of a dye-nucleotide conjugate, and should be more efficient and uniform.

Here are a couple of suggestions:

1) cDNA may have been lost prior to labeling. Add 1 µL of glycogen (molecular biology grade), containing 10–20 µg, to the cDNA before precipitating it with ethanol.

2) Make sure to add sodium acetate as the salt and not ammonium acetate. After pelleting the cDNA, resuspend it in 5 µL water.

3) If generating long cDNAs, it will help to heat-denature the sample. Heat it at 95°C for 5 minutes and then put it on ice for a few minutes. Then centrifuge it for a few minutes just prior to the labeling reaction.

4) You want the tube to be at room temperature for the labeling reaction. Add the 3 µL of buffer and mix it in. Then add the dye and vortex it vigorously for at least 15 seconds.

The ULYSIS® oligonucleotide chemical labeling reagents allow you to rapidly and easily couple our fluorescent dyes to purine bases in nucleic acid polymers. The method, the Universal Linkage System (ULS™), is based on the use of a platinum dye complex that forms a stable adduct at the N7 position of guanine. ULYSIS® labeled probes are used in fluorescence in situ hybridization (FISH) and numerous other applications.

We recommend using ethanol precipitation to concentrate the sample first to calculate the DNA concentration and degree of labeling.

After labeling and purifying your DNA, you can add 10 µg of salmon sperm DNA. This should improve your recovery after precipitation.

An oligonucleotide labeled with a ULYSIS® Nucleic Acid Labeling Kit should survive 100°C for 5 minutes, and storage at 68°C overnight should also not cause any dissociation of the complex.

It might be possible to label larger probes with the ULYSIS® Nucleic Acid Labeling Kits, but the dye will likely need to be diluted to avoid (or at least reduce) problems with aggregation. 

A preliminary protocol modifies our DNA-labeling protocol: Do not nuclease-treat the RNA, but label it directly by incubating for 10 minutes at 90°C or 15 minutes at 85°C. Add 2 μg of glycogen for every 1 μg of RNA and purify by ethanol precipitation. Refer to these publications:

• Babak T, Zhang W, Marros Q et al. (2004) Probing microRNAs with microarrays: tissue specificity and functional inference. RNA 10(11):1813–1819.
• Hiley SL, Jackman J, Babak T et al. (2005) Detection and discovery of RNA modifications using microarrays. Nucleic Acids Res 33(1):e2.
• Torchet C, Badis G, Devaux F et al. (2005) The complete set of H/ACA snoRNAs that guide rRNA pseudouridylations in Saccharomyces cerevisiae. RNA 11(6):928–938.

Long-term storage for the ULYSIS® labeled probes can be done in just about any kind of buffer, TE, formamide, hybridization buffer, or ethanol. We suggest using your normal storage conditions as long as you protect the probes from light. ULYSIS® conjugates are very stable.

Yes, there are numerous examples of ULYSIS® labeled probes that have been used in microarray analysis. Here are a few publications for your reference:

• Babak T, Zhang W, Marros Q et al. (2004) Probing microRNAs with microarrays: tissue specificity and functional inference. RNA 10(11):1813–1819.
• Hiley SL, Jackman J, Babak T et al. (2005) Detection and discovery of RNA modifications using microarrays. Nucleic Acids Res 33(1):e2.
• Torchet C, Badis G, Devaux F et al. (2005) The complete set of H/ACA snoRNAs that guide rRNA pseudouridylations in Saccharomyces cerevisiae. RNA 11(6):928–938.

These probes are not subject to radiolysis like radiolabeled probes; therefore, you can store them for at least one year.

It can be used to label single-stranded antisense RNA for northerns, Southerns, or in situ hybridization. The same kit can also be used to label PCR products, plasmids, gel-purified cDNA inserts, and oligonucleotides for northern and Southern blotting and in situ hybridization. Sensitivity is comparable to that obtained with 32P-radiolabeled probes and enzymatically synthesized biotin-labeled probes when used with the Ambion® BrightStar® BioDetect™ Kit for nonisotopic detection and BrightStar®-Plus Positively Charged Nylon Membranes.

1. Sample purity:

• There should be no free nucleotides in the preparation (e.g., from PCR).
• RNA samples must be free of DNA, e.g. a transcription template.
• Samples must be free of proteins.
• Nucleic acid solutions should be free of phenol or alcohol contaminants, the pH should be between 2.5 and 10, and the salt concentration should be less than 20 mM.

2. The sample concentration should be 0.5–50 ng/μL.

To determine the current specific activity of your radiolabeled probe, use the following calculation: cpm/µL = original cpm/µL/2^(days after original reading/14.3)