Hi all,
I need help calculating the melting temperature (Tm) of barcoded primers for PCRs. I am using two barcoded primers to separate dozens of samples' DNA sequences.
Using the equation Tm=2(A+T)+4(C+G), non-barcoded primers have Tm's of 60C and 62C. However, inclusion of the 6-nucleotide barcode as well as a 2-nucleotide pad raises the Tm to over 80C.
If it's only the primers that anneal to targeted sequence, can I ignore the nucleotides of the barcodes and pads, and just use the Tm of non-barcoded primers?
Yes, since the barcode does not anneal to the template it should not be part of the Tm estimation. However, you need to check that your barcode DNA does not anneal for this to be true. Furthermore, you also need to check that your barcode does not anneal to any other barcode or primer part(s), forming hairpins, polymers etc. Assuming you have many, this becomes complicated. Finally, for more precise Tm estimation the nearest-neighbor model is often used, which also takes salt concentration into account as that also affects Tm. You could try our web tool PrimerDesign-M, which does all the above automatically, and see if that works for you. See https://www.hiv.lanl.gov/content/sequence/PRIMER_DESIGN/primer_design.html
the equation that you quote is usable but only for short primers of less than about 14 bases. .Better to use one of the on line calculatore based more on nearest neighbour bases or Tm (°C) = 81.5 + 0.41(%GC) - (675/N) where %GC is the percentage of G and C nucleotides in the oligo and N is the length of the oligo if your primers are longer than this , You should use the annealing temperature appropriate to only those bases annealing in the first cycles of pcr. The barcode will be incorporated into the product after the first cycle and then the whole primer can anneal but if you include this sequence in the annealing temperature for the first cycle the primer may never anneal and the pcr will fail
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