I have a fwd primer with 37% GC content and the rev primer with 61% GC content. The Tm of the fwd primer is 55.6 C and that of the rev is 58.2 C. Upon performing gradient PCR, I saw bands at 50C and 53C but not at 52 and 54C. When I performed PCR at 50C, I did not get any bands. I am considering using DMSO to lower the Tm of the second primer, but am not sure if this could be a reason I am not getting any bands. What problems may arise from differences in GC content between primers, and could this be a reason I am getting sporadic results?
GC bonds are not just stronger than AT bonds. They're also less specific. GC-rich regions are much more prone to mis-pairing with other GC-rich regions, than balanced regions are. This gives you false bands, and inconsistent results due to secondary structures. Due to this intrinsic lack of specificity, you should avoid GC-rich regions during any kind of molecular work, wherever possible.
If you do not have the freedom to change your primer away from the GC-rich region, then yes, it's a good idea to try additives like DMSO and betaine ("GC enhancer"). These can often mitigate the problem, and in rare cases even make an all-or-nothing difference.
GC bonds are not just stronger than AT bonds. They're also less specific. GC-rich regions are much more prone to mis-pairing with other GC-rich regions, than balanced regions are. This gives you false bands, and inconsistent results due to secondary structures. Due to this intrinsic lack of specificity, you should avoid GC-rich regions during any kind of molecular work, wherever possible.
If you do not have the freedom to change your primer away from the GC-rich region, then yes, it's a good idea to try additives like DMSO and betaine ("GC enhancer"). These can often mitigate the problem, and in rare cases even make an all-or-nothing difference.
I would suggest to use annnealing just near about your highest melting temperature of primer set 1-2 degree lower as well as higher than highest one. It works well with these type of primer sets try the 60-63 degree annealing temperature for your PCR it will work.........try it.........otherwise as suggested by John Schloendorn you have to redesign or use alternative methods...........
Best way to redesign your primer and set GC contain around 50-60% for both the primers. and based on primers set your TM value in PCR.
1. The general suggestion of GC content is between 40-60 %. Your 2 primers are a little bit fall out of the range.
2. Check the tendency of your primers to form secondary structures ('hairpins', self-dimer, cross-dimer). They adversely affect primer/template annealing and thus the amplification. They greatly reduce the availability of primers to the reaction.
Thank you all for your suggestions.
1. Redesigning the primer was not suitable since I was working on amplifying a GC-rich region.
2. I repeated the experiment and got a faint band at the desired position when I performed PCR at 50C. I excised this band and performed gel elution, then used this as the template and performed PCR again using the same primers and at the same temperature, and was able to get good bands from then on. I sequenced the PCR product and found that it matches my target.
I suspect that my problem arose because of nonspecific binding in the genomic DNA. As indicated by John Schloendorn, GC-rich primers often mispair with other GC-rich regions. When I used the PCR product as the template, the pairing region on the template was narrowed down and the primers bound to the template with more specificity.
Once again, thank you for all your very informative suggestions.
I am having somewhat similar issue. , confused about the Tm as I design it once while the company send me very different Tm values. So m stuck what to choose. I have a lot of sequences to analyse and limited time
Hi,
I have found that the Promega Tm calculator and Thermo multiple primer analyzer give good estimates of Tm. In any case, you will need to test +/- 3-5 Celsius from the bioinformatics estimate.
Best,
Subramaniyam
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