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Labeling of Nucleic Acids


Two important considerations in determining what labeling system will work best in a given application are: (1) size and type of the nucleic acid to be labeled; (2) choice of label required for the application.

Size and type. Longer strands of DNA (>100 bp) or circular DNA (i.e. plasmid DNA), RNA or PNA (peptide nucleic acid) used in blots, in situ hybridization, subtractive hybridization, or other procedures are efficiently and reliably labeled with the PHOTOPROBE® labeling reagents or the FastTag® Nucleic Acid Labeling System. Either labeling method results in the covalent coupling of the label to the sample. These systems ensure multiple site labeling over the entire length of the nucleic acid resulting in greater accessibility of the affinity tag or greater sensitivity in detection.

The integrity of the nucleic acid is preserved in this non-destructive reaction making it useful for applications where it is necessary to use the intact, original sample. With the PHOTOPROBE® or FastTag® labeling systems, the entire length of the original nucleic acid sample, rather than copies, is directly, labeled. In contrast, enzymatic labeling methods such as random priming or nick translation are difficult to control and don’t label the original nucleic acid sample. Instead, these methods result in a labeled copy produced from the original template.


Thus, the PHOTOPROBE® or FastTag® chemical labeling method is especially convenient for labeling samples that will be used to observe cellular localization of nucleic acid (e.g. plasmid DNA in gene delivery or siRNA) or quantitative comparison.

Shorter strands of nucleic acids such as oligonucleotides, PCR primers, or capture probes used to identify nucleic acid binding proteins are specifically and efficiently labeled at either the 5’ or the 3’ end using the 5’ E’ ndTag™ or the 3’ E’ ndTag™ Nucleic Acid Labeling Systems, respectively. The 5’ E’ ndTag™ or the 3’ E’ ndTag™ Kits can be used to attach a single fluorochrome or affinity tag at the appropriate end of nucleic acids. 5’ E’ ndTag™ Labeling kit uses both DNA and RNA as a substrate whereas the 3’ E’ ndTag™ Kit will selectively label only DNA.

Application. The second important factor in determining the most appropriate labeling system is the choice of label, hapten, or affinity tag required for the given application. The PHOTOPROBE® reagents incorporate a specific tag (e.g. biotin) into nucleic acid in one simple step. The versatility of the FastTag® Labeling kit, 5’ EndTag™ or 3’ EndTag™ Labeling Kits allows a variety of tags to be incorporated. Some commonly used labels that are available from Vector Laboratories are briefly described as follows. Detection reagents for these labels are listed on page 10.

Choose a Labeling System based on Nucleic Acid Type and Size

Choose a Labeling System

 

Biotin - Because of the extraordinary affinity of avidin and streptavidin to biotin and the many biotin-avidin/streptavidin systems available, this label is ideal for a variety of applications including in situ hybridization, blotting, and affinity binding.

DNP (Dinitrophenyl) - Because DNP is not found endogenously in tissue, it is an excellent alternative to biotin. High affinity, purified antibodies are available for detection or amplification of the signal.

Fluorescein - This fluorescent label can be directly detected or used as a hapten and detected with our high affinity antibody reagents. (Excitation maximum at 495 nm; emission maximum at 515 nm).

Fucose - This unique label is ideal for reversible binding of labeled nucleic acid to the matrix, VECTREX® AAL. Fucose labeled nucleic acids can be bound and eluted under mild conditions. (For more information, please see page 17). Alkaline phosphatase Aleuria aurantia lectin can be used in dot blot applications to assess labeling efficiency with the fucose label.

Texas Red® - This fluorescent label is a high quantum yield rhodamine derivative that can be directly visualized. The label can also be detected, and the signal amplified, using our affinity-purified antibody conjugates to rhodamine. (Excitation maximum at 595 nm; emission maximum at 615 nm).

Choose a Labeling System based on Application and Label

Choose a Labeling System

PHOTOPROBE® Reagents and the FastTag® System

PHOTOPROBE® Reagents and the FastTag® Nucleic Acid Labeling Systems are the methods of choice for incorporating a label at multiple sites along the entire length of the nucleic acid. The labeling reaction does not destroy the original nucleic acid nor creates its copy. The integrity of the original sample is preserved. Single-15, 28 or doublestranded11,20 DNA, circular DNA, 45, 58 RNA, 22 siRNA, 49 or PNA (peptide nucleic acid) can be labeled with the same reagents.

Labeling with either system is based on aryl azide chemistry in which either reagent, when exposed to heat or light, becomes activated and incorporates into the nucleic acid without base specificity. The labeling reaction can be carried out using a mercury vapor bulb (sun lamp), a UV lamp which produces light in the 350 nm to 370 nm range, a halogen lamp, a heating block, or a thermal cycler providing the investigator with great flexibility in experimental design.

A distinct advantage of the simple setup in each of these labeling options is the ease and economy of scaling up.

PHOTOPROBE® Biotin incorporates biotin in one reaction step. PHOTOPROBE® (Long Arm) Biotin has an extra long linker arm and should be used if increased distance between the sample and the tag is needed. In a similar manner, PHOTOPROBE® Amine incorporates primary amines into nucleic acids. These amino groups can subsequently be used to attach haptens, affinity tags, or fluorochromes, or to immobilize nucleic acids to a solid matrix.

The FastTag® Nucleic Acid Labeling System utilizes a disulfide-containing universal linker that is incorporated into nucleic acid simply by using heat or light. Reducing the disulfide bond yields a free thiol group. A variety of thiol-reactive haptens, fluorochromes, affinity ligands or other markers can then be covalently bound to the nucleic acid via the FastTag® reagent thiol group. See page 7 for a list of thiol-reactive reagents available from Vector Laboratories.

 

PHOTOPROBE® Labeling Reaction

PHOTOPROBE® labeling (above) occurs in one short heat- or photo- labeling step.

FastTag® labeling (right) occurs in three easy steps:

Step 1. The FastTag® Universal Linker is incorporated into the nucleic acid upon exposure to heat or light

Step 2. The disulfide bond is reduced, yielding a free thiol group

Step 3. A covalent bond is formed between the FastTag® reagent thiol and a thiol-reactive hapten, fluorochrome, affinity ligand, or other marker.

FastTag®Labeling Reaction

Each PHOTOPROBE® Biotin Kit contains the following components to label up to 250 µg of nucleic acid (or to carry out up to 50 labeling reactions):

  • PHOTOPROBE® Biotin Reagent
  • Tris Buffer
  • sec-Butanol
  • Biotinylated DNA Standard
  • Precipitant


Labeling Systems

FastTag® Labeling Kit MB-8000 • 1 Kit 
PHOTOPROBE® Biotin SP-1000 • 0.5mg 
PHOTOPROBE® (Long Arm) Biotin SP-1020 • 0.5mg 
PHOTOPROBE® Biotin 
Labeling and Detection System S 
PK-1906 • 1 Kit 
PHOTOPROBE® Amine SP-1070 • 0.5mg


The FastTag® Labeling Kit† contains the following reagents to label up to 250 µg of nucleic acid (or to carry out up to 50 labeling reactions):

  • FastTag® Reagent
  • Tris Buffer
  • Citrate Buffer
  • Reducing Reagent
  • sec-Butanol
  • Precipitant


†Use this kit with any thiol-reactive label.

The PHOTOPROBE® Amine Kit includes the following components to label up to 360 µg of nucleic acid (or to carry out up to 72 labeling reactions):

  • PHOTOPROBE® Amine
  • Borate Buffer
  • sec-Butanol
  • Precipitant

 

Thiol-reactive Labeling Reagents

Biotin Maleimide SP-1501 • 12mg
DNP Maleimide SP-1503 • 1mg
Fluorescein Maleimide SP-1502 • 12mg
Fucose Maleimide SP-1504 • 500µg
Texas Red® Maleimide SP-1505 • 3.6mg

These thiol-specific labels can be used with the FastTag® Labeling Kit, 5’ EndTag™ or 3’ EndTag™ Labeling Kits.

Aryl Azide Chemistry

5’ EndTag™ and 3’ EndTag™ Labeling Systems

End labeling is a favored method for applications where an internal label might interfere with hybridization or sequence-specific protein binding. Short oligonucleotides are labeled more efficiently with these systems than with other methods. In addition, end labeling of oligonucleotides is an economical alternative to having labels inserted during synthesis.

Both the 5’ EndTag™ and the 3’ EndTag™ Nucleic Acid Labeling Systems enable the covalent attachment of a variety of fluorescent dyes, haptens or affinity tags to the respective ends of the nucleic acids using thiol-specific chemistry.

5’ EndTag™ is ideal for labeling PCR primers because a label is attached only at the 5’ end, leaving the 3’ end available for polymerization. The end position of the label generally does not interfere with hybridization or nucleic acid binding and is, therefore, appropriate for binding of capture probes to affinity matrices30, 52 and for electrophoretic mobility shift assays (EMSA). 19, 37

The 5’ EndTag™ system labels 5’ ends of DNA, RNA, 30, 60 or unmodified oligonucleotides. 47, 57

3’ end labeling is preferred over 5’ end labeling if the terminal phosphate at the 5’ end must be preserved. 3’ EndTag™ labeled nucleic acids can be used for applications such as DNA hybridization, PCR, in situ hybridization, or EMSA. The 3’ EndTag™ system enables simple and uniform labeling of 3’ ends of DNA.

5’ EndTag™ Kit MB-9001 • 1 Kit
3’ EndTag™ Kit MB-9002 • 1 Kit

A thiol-reactive label is not included in the kit, but one can be selected from the list below.

Thiol-reactive Labeling Reagents

Biotin Maleimide SP-1501 • 12mg
DNP Maleimide SP-1503 • 1mg
Fluorescein Maleimide SP-1502 • 12mg
Fucose Maleimide SP-1504 • 500µg
Texas Red® Maleimide SP-1505 • 3.6mg

 

Transmission electron micrographs

Transmission electron micrographs of molecules of influenza A viral ribonucleoprotein particles (vRNPs) labeled at the 5’ end of the vRNA with biotin using the 5’ EndTag™ Kit, and further labeled with streptavidin gold. vRNPs from influenza A were purified according to Kemler et al., (1994 Virology, 202:1028- 1033) on a glycerol gradient with modifications as described in Wu et al., (2007, Virol J. Jun 4;4:49). Courtesy of Drs. Winco WH Wu and Nelly Panté, University of British Columbia, Vancouver BC, Canada.

Nuclear import assay

Nuclear import assay in digitonin-permeabilized HeLa cells of biotinylated vRNPs. vRNPs were labeled first at the 5’ end of the vRNA with biotin using the 5’ EndTag™ Kit, and then with Vector® Fluorescein Streptavidin. This allowed for direct fluorescence visualization of the vRNPs on a confocal fluorescence microscope. Nuclear import assays were carried out as described in Wu et al., (2007, Virol J. Jun 4;4:49). The negative control consists of vRNPs added to the cells in the absence of energy and exogenous cytosol. In the presence of energy and cytosol, the fluorescein-labeled vRNPs successfully enter the nucleus, with a high degree of nucleolar staining. Courtesy of Drs. Winco WH Wu and Nelly Panté, University of British Columbia, Vancouver BC, Canada.

5’ End Labeling Kit

Labeling with the 5’ EndTag™ Kit is achieved by a simple two step process. The first step is to incorporate a thiol group onto the 5’ end of the oligonucleotide. This is accomplished by T4 polynucleotide kinase which transfers a thiolphosphate from ATPγS to the 5’-OH group of the oligonucleotide. In the second step the thiolcontaining oligonucleotide is incubated with a thiol-reactive label (e.g. biotin-maleimide). Labeling requires about 1 hour with very few minutes of hands-on time.

The 5’ EndTag™ Kit is designed to perform 10 labeling reactions of up to 0.6 nmols of 5’ ends (e.g. about 5 µg of a 25 base oligo) per reaction and includes:

  • T4 polynucleotide kinase
  • 10x reaction buffer
  • ATPγS
  • Precipitant
  • Alkaline Phosphatase

(A thiol-reactive label is not included.)

Figure A schematically shows the 3’ EndTag™ labeling reaction. Figure B shows the labeling reaction for the 5’ EndTag™ Labeling System.

3’ End Labeling Kit

Labeling using the 3’ EndTag™ Labeling Kit is achieved in two steps. The first step is to incorporate a thiol group onto the 3’ end of the DNA. This is accomplished by using a terminal transferase enzyme (TdT) to attach SH-GTP, a modified guanosine triphosphate containing a thiol, to the double or single stranded DNA end. Blunt, overhanging, or recessed 3’-OH ends may also be used. In the second step the thiol-containing oligonucleotide is incubated with a thiol-reactive label (e.g. biotin maleimide). Labeling time is about 1 hour with very little hands-on time. Labeling efficiency is equivalent to that achieved with traditional methods but the 3’ EndTag™ labeling system offers greater versatility in the choice of label at a fraction of the cost.

The 3’ EndTag™ Labeling Kit is designed to perform 20 labeling reactions of up to 0.5 nmols of 3’ ends (e.g. about 3.3 µg of a 20 base oligonucleotide) per reaction and includes:

  • Terminal transferase (TdT)
  • SH-GTP
  • 10x TdT buffer
  • Precipitant

(A thiol-reactive label is not included.)

A thiol-reactive A thiol-reactive b