Fluorescent Tagging of Primers
Fluorescent tagging has historically been used to label proteins. As such, there are cheap commercial means to obtain fluorescently tagged proteins and also efficient ways of tagging proteins in the lab.[1–3] However, primers can also be fluorescently tagged for use in PCR. There are a range of tags to be used depending on the desired color.
1.6-FAM = blue; the most intense dye
2.HEX = green
3.NED = yellow; replacement for TET/TAMRA
4.ROX = red; used as a size standard label
5.TAMRA = red or yellow; can be an alternative size standard label
6.TET = yellow; least intense dye and has strong overlap with 6-FAM
Despite the range of options, ordering fluorescently tagged primers can be expensive. For example, ordering them from Integrated DNA Technologies will cost upwards of $80 per primer. [4] The price is even more egregious from places like Sigma Aldrich. Furthermore, if one wanted to set up a molecular beacon by tagging one end with a primer and the alternate end with a quencher, that would cost even more. For example, ordering an oligonucleotide with the dark quencher, Dabcyl, at the 3’ end costs $80 at the cheapest. It is then easy to imagine how expensive ordering an oligonucleotide with both a 5’ fluorophore and a 3’ quencher.
The alternative is to add a fluorescent tag ourselves after ordering the primers.[5] This has been historically done by first adding phosphoramidite and then adding a dye-NHS ester. However, companies like ThermoFisher now sell all-in-one reagents that complete the tagging reaction in one step. However, these are likely to cost upwards of $300.[6] The cost is not far off from the traditional method of purchasing the two reagents separately.
Given these prices, I think it is more financially advantageous to explore dye-based qPCR methods.[7] In these methods, a dsDNA binding dye such as SYBR Green is used. “The dye displays weak background fluorescence that increases dramatically upon binding to dsDNA. Thus, amplification of the target sequence results in an increase of fluorescence that is directly proportional to the amount of dsDNA present at each PCR cycle. This type of qPCR assay requires only two sequence-specific primers, making it a rapid and cost-effective way to interrogate a large number of samples/targets.”
One disadvantage of intercalating dye-based methods is that they detect any dsDNA produced in the reaction. This includes off target amplification products and primer-dimers, which results in inaccurate quantification.” One way to verify the results would be to perform a denaturation curve to verify reaction specificity.
1. Galbán J, Andreu Y, Sierra JF, Marcos S de, Castillo JR (2001) Intrinsic fluorescence of enzymes and fluorescence of chemically modified enzymes for analytical purposes: a review. Luminescence, 16(2):199–210. https://doi.org/10.1002/bio.633
2. Liu H-W, Chen L, Xu C, Li Z, Zhang H, Zhang X-B, Tan W (2018) Recent progresses in small-molecule enzymatic fluorescent probes for cancer imaging. Chemical Society Reviews, 47(18):7140–7180. https://doi.org/10.1039/C7CS00862G
3. Toseland CP (2013) Fluorescent labeling and modification of proteins. Journal of Chemical Biology, 6(3):85–95. https://doi.org/10.1007/s12154-013-0094-5
4. Fluorophores. https://www.idtdna.com/site/Catalog/Modifications/Category/3
5. Giusti WG, Adriano T (1993) Synthesis and characterization of 5’-fluorescent-dye-labeled oligonucleotides. Genome Research, 2(3):223–227. https://doi.org/10.1101/gr.2.3.223
7. Dye-based qPCR & RT-qPCR | NEB. https://www.neb.com/applications/dna-amplification-pcr-and-qpcr/qpcr-and-rt-qpcr/dye-based-qpcr