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	DNA/RNA Labeling

All of Vector’s nucleic acid labeling kits are non-radioactive and offer many advantages over radioactivity, including excellent sensitivity in a shorter period of time; prolonged stability of reagents and labeled probes; and choices of detection and affinity binding matrices. Vector’s labeling systems employ different approaches which (1) incorporate the label at multiple sites along the entire length of the nucleic acid, or (2) incorporate the label only at the 3’ or 5’ end.

For those new to the field or those seeking straightforward, cost effective approaches in optimal labeling and detection of DNA/RNA probes please see the following Molecular Biology Guide. Instructive diagrams showing labeling methodologies are accompanied by photographic examples submitted by independent investigators that highlights their recent research. Practical insight is offered into choice of labeling method based on probe size, type, application and tag. Subsequent detection of the labeled probes using chromogenic or fluorescent visualization is described for various applications including microarrays, blotting and in situ hybridization. Step-by-step protocols and referenced publications make this a valuable and contemporary reference tool. You may download a copy here or request a free copy on our Catalog & Brochures request page under the Support menu.

MBB.pdf (PDF)

Choosing a Labeling System

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 of nucleic acid. 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 and interactions of nucleic acid with cytoskeletal components (e.g. plasmid DNA in gene delivery) 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’ EndTag™ or the 3’ EndTag™ Nucleic Acid Labeling Systems, respectively. The 5’ EndTag™ or the 3’ EndTag™ Kits can be used to attach a single fluorochrome or affinity tag at the appropriate end of nucleic acids. 5’ EndTag™ Labeling kit uses both DNA and RNA as a substrate whereas the 3’ EndTag™ Kit will selectively label only DNA.

References:

FTrefs06-10.pdf (PDF)

PPBrefs9_09.pdf (PDF)

5ETrefs07-10.pdf (PDF)

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.