There is a lot of information that goes into understanding DNA and RNA radiolabeling. Radioisotope labeling is a traditional method for nucleic acid labeling, and it is highly essential to the detection, visualization, and purification of DNA and RNA.
Reasons for DNA and RNA Radiolabeling
Radiolabeled nucleotides typically detect specific nucleic acid sequences. By incorporating radiolabeled nucleotides enzymatically, DNA and RNA sequences are detected and analyzed. Depending on the type of application, DNA and RNA sequences are labeled at the 5' or 3' end. A few of the typical reasons for radiolabeling include:
- To generate information on gene integrity and copy number (blot).
- To diagnose specific sequences and chromosomal aberrations (in situ hybridization).
- To simultaneously measure the relative expression of RNAs (microarray analysis).
- To discover protein-nucleic acid interactions (electrophoretic mobility shift assays or FRET).
Choosing a Radiolabeling Method
When choosing a labeling system, consider the size and type of nucleic acid you're working with. Large DNA, plasmid DNA, and RNA for blots and in situ hybridization can be labeled throughout by random incorporation of a covalently coupled label. While covalent probes produce excellent sensitivity, enzymatic methods are widely available in labs, making them a popular choice.
Before choosing a method, you should also think about the application you'll be using your labeled nucleic acid in. For protein interaction studies, large fluorophore labels can interrupt interactions due to their steric hindrance and chemical properties. Smaller labels or labeling a site that is farther away from the interaction location will avoid this. If you're looking for signal amplification, secondary reporters such as antibody conjugates like avidin/streptavidin are an excellent choice.
Common Methods
- Random Primer
- Nick Translation
- Klenow
- Single Nucleotide Terminator Labeling
- DNA 3' End and DNA 5' End Labeling
- EDC Labeling
While there is much more that goes into DNA and RNA radiolabeling, knowing the reasons for radiolabeling nucleic acids, understanding how to choose a method, and familiarizing yourself with common methods are the basis for understanding DNA and RNA radiolabeling. Visit Moravek for more information on radiolabeling services.