Dear all, I have found that perfect dinulceotides repeats (CA/GA) need in general a higher annealing temperature than imperfect motif (e.g. (GT)12(GA)17GCAAA(G)3). Is it a rule or an exception for plants? It could be related with the palindromic nature of dinucleotides motifs? Can someone recommend some papers or lectures? Thanks
I’m not sure that the palindromic nature of dinucleotides motifs would affect the annealing temperature, unless it causes difference in the melting temperature of the targeted DNA sequence. However, basically optimal annealing temperature is related mainly with the melting temperatures of the less stable primer-template pair and the product length (please see Rychlik’s article: Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res. 1990 Nov 11; 18(21): 6409–6412).
In the other hand, you may find some explanation of your findings if considering the significant differences in the number and size of the perfect and imperfect motifs in plants. You may find further information about that in this article: “An empirical review: Characteristics of plant microsatellite markers that confer higher levels of genetic variation. Appl Plant Sci. 2015 Aug; 3(8): apps.1500025.”
I hope I was useful in sharing answering your questions.
Hi Abdul, when I mentioned any probably relationship between palindromic nature of dinucleotides motifs and the annealing temperature, I was thinking in the raise of temperature to increase the especificity of union to obtain interpretable amplification products. Considering the palindromic structure of perfect dinucleotides-microsatellites, could it be possible that the template be folded during amplification before the union dNTPs to it more easily than the most complex repeats might do? If it's the case, then markers formed by perfect repetitions would need higher annealing temperatures; isn't it? Thanks for your comments and the references. About it, I couldn't find the Rychlik paper, would you be so kindly to send me it privately? Here is my email: [email protected]
Regards
Hi Ricardo, I thank you for this clarification. Your thinking about the possible quick folding of the of perfect dinucleotides-microsatellites due to its palindromic structure is very logic and I completely agree with you. Just I would like to add that the forming of such secondary structure of the DNA template could promote "polymerase jumping" during PCR amplification, which yields amplicons with sizes different than the expected ones. So, raising annealing temperature could also avoid this phenomena, and the yielded amplification products become more reliable for the correct interpretation of the results.
I have sent you the requested paper on your email.
regards
- Badges
- Science topic
- Similar topics
- Papers