Hello,
I identified the +1 of a given transcript using classical primer extension with radioactivity. However, the procedure is arduous, especially because most of people of my lab cannot work with radioactivity. It is why we are trying to find an alternative way, such fluorescence primer extension. Using the same primer used for the first primer extension (but labelled with FAM-6) we obtained a very clear peak, but 5 bp downstream of the first +1. I do not know why it may happen. Maybe altered electrophoretic mobility? I will add some controls to check this point, but if someone has expertise in this field I would thank any orientation.
Thanks
Compare to a radionuclide, 6-Carboxyfluorescein is a large compound and it should not be surprising if the electrophoretic mobility is altered.
Digoxigenin labeled cRNA molecules behaved similarly in my hands.
Agreed, but then I would expect that it would behave as a larger cDNA molecule instead a shorter one as it occurs. Maybe the 6-FAM charge makes the cDNA migrate faster?
Are you using capillary electrophoresis, instead of gel electrophoresis?
I could find a few reports stating that apparent sizes of 6-FAM-labelled nucleotides are smaller on capillary electrophoresis.
http://people.ibest.uidaho.edu/~foster/Papers/9603.pdf
https://www.ncbi.nlm.nih.gov/pubmed/11545394
The abstract of the second paper also states that the apparent size discrepancy is smaller on classical urea/polyacrylamide gels.
I hope these give some helps.
Interaction of DNA with other molecules always has some influence on the physicochemical properties, including mobility on gels. Even intercalating EtBr, a commonly used fluorophor causes larger fragments of DNA to run slightly faster. You can test this by running too halves of a gel at the same time, one half containing EtBr and the other without the dye and stain this half with EtBr after the run - now compare the mobilities!
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