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™ 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™ 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. 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™ Labeling Kit
The 5’ EndTag™ System labels 5’ ends of DNA, RNA, or
unmodified oligonucleotides. 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.
Labeling with the 5’ EndTag™ 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™ 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.
|