What are the different labeling techniques I could use to make probes labeled with CyDye?
There are a number of different routes to produce probes labeled with fluorescent dyes, the most utilized are: 1. First-strand cDNA labeling: Involves direct incorporation of the CyDye-labeled nucleotide into the cDNA using a reverse transcriptase enzyme. This is the preferred route for most customers, the draw back has been the low incorporation rate of the CyDye-labeled nucleotides by the enzymes. (i.e. number of dye molecules that can be incorporated and detected). Most enzymes do not incorporate Cy3 and Cy5 dyes evenly; hence, the signals generated on hybridization are not usually even. Cy5 is usually not incorporated as well as Cy3; however, the CyScribe enzyme in the CyScribe First Strand cDNA Labeling Kit demonstrates a high rate of incorporation of both Cy3- and Cy5-labeled nucleotides. 2. Post labeling: This method involves incorporating a chemically reactive nucleotide analog (amino allyl-dUTP) in to first-strand cDNA during synthesis. The analog incorporates similarly to an
