Hi!
I am to site-specifically label dsDNA parts using PCR with fluorescently labeled primers. For my objective it would be optimal if the primers could carry the fluorophore as close to their 3'-end as possible, preferably exactly at the 3'-end. I am worried however that perhaps this would prevent the polymerase's ability to elongate during PCR.
I have seen others label the primers as close as 1 position upstream of the 3'-end e.g. in:
Nazarenko I, Lowe B, Darfler M, Ikonomi P, Schuster D, Rashtchian A. Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore. Nucleic Acids Res. 2002 May 1;30(9):e37. doi: 10.1093/nar/30.9.e37. PMID: 11972352; PMCID: PMC113860.
Can DNA polymerase elongate if the 3'-end of the primer is modified with a fluorescent tag?
Thankful for any input!
All the best,
Niklas Eckert Elfving
Uppsala University
If your 3'-end nucleotide is T, for example, you can add fluorophore to the thymine circle.
IDTDNA manufactures oligos with FAM and fluorescein at the 3'end base so they must be fairly certain that pcr will work but an email to IDTDNA technical support would be useful. They also have a range of linkers to keep the fluorophore distant from the dna base
https://eu.idtdna.com/site/Catalog/Modifications/Dyes
Thank you both for your suggestions. For now I have ordered an oligo labeled with Cy5 one base upstream of the 3’-end. I Will contact IDT as you propose Paul and if it makes sense further down the line I Will try it out with the label at the 3’-end. I Will update this thread with the outcomes.
Thank you for answering Ruslan!
I know that Cy3 can be added to DNA:s by PCR when located as far up as 13 bp from the 3’-end on the primers but I need another fluorphore such as Cy5 for it to be compatible as FRET acceptor and closer to the 3’-end to be in the dynamic range for FRET. If I understand you correctly you are opposing the possibility to incorporate Cy5 by PCR when located close to the 3’-end of the primer But not necessarily any fluorphore? Have you incorporated other fluorphores than Cy5 close to the 3’-end?
Best regards,
Niklas
Yes the efficiency of the PCR is my concern. The fluorophore donor Will be located on a transcription factor peptide that Will bind to a transcription factor motif in the DNA part that we want to label by the fluorogenic PCR. The primers Will anneal adjacent to the transcription factor motif and we thus want the fluorophore acceptor to be as close to the 3’-end of the primer as possible. Since we are labeling the peptide with TMR we are limited to TMR compatible acceptor fluorphores. You are however probably right in that the PCR Will be of very low efficiency or not work at all with a to big fluorophore to close to the 3’-end.
Hi!
As an update to the thread I can say that we have tried the site specific fluorophore labeling by PCR using both an internal Cy5 on the fourth position and with a fluorescein-dT base as the fourth base of the reverse primer (counting from the 3'-end). The Cy5 incorporation was not successful neither by PCR using Q5 polymerase nor by Klenow extension of PCR product. The primer carrying a dT-Fluorescein as the fourth base on the other hand worked great in Q5 PCR. We have not yet tried the Fluorescein primer in Klenow extension but will in the near future.
So Ruslan you were indeed right about the polymerase not being capable of ignoring Cy5 so close to the 3'-end!
Thank you all for your answers and suggestions!
All the best,
Niklas