End labeling is a favored method for applications where an internal label might interfere with hybridization or sequence specific protein binding.
5' or 3' EndTag[TM] labeled nucleic acids can be used for applications such as DNA hybridization, PCR, In-situ Hybridization, the binding of capture probes to affinity matrices, or electrophoretic mobility shift assays (EMSA). 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[TM] and the 3' EndTag[TM] 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. Labels containing thiol-reactive groups (maleimides, iodoacetamides, etc.) can easily be incorporated.
Labeling time is about 1 hour with very little hands-on time.
5' EndTag[TM] Labeling Kit
The 5' EndTag[TM] System labels 5' ends of DNA, RNA, or unmodified oligonucleotides. 5' EndTag[TM] is ideal for labeling PCR primers because a label is attached only at the 5' end, leaving the 3' end available for polymerization.
Labeling with the 5' EndTag[TM] Kit is achieved in two steps:1. T4 polynucleotide kinase transfers a thiophosphate from ATPγS to the 5' -OH group of the nucleic acid. (5' phosphorylated ends are converted to 5' -OH groups with the included alkaline phosphatase).
2. Thiolated sample is coupled to a thiol-reactive label. The 5' EndTag[TM] 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. Thiol-reactive label is not included in the kit and should be selected separately.