Hello everyone,
I amplified from cDNA template my sequence of interest using SuperFi II polymerase and I obtain two bands, one at 1300bp (the weight that i want to have) and other strong band at 600bp.
I decided to cut and purify my band to re-amplified it with primers to add blunt ends in order to clone it by Gibson assembly and... I got again the 600bp band!! And, in a lesser way, a 1300bp band. ( I tried to directly amplify from first cDNA template with primers to add blunt ends, but i only obtained the 600bp band...)
Does anyone know what is going on?
Thanks in advanced to everyone!
Best guess: there is a site somewhere within your 1300bp amplicon that weakly binds one of your primers.
A key thing to remember about PCR is that every cycle incorporates more primer sequence into the amplicon population, so any given mismatch event only needs to happen once. After that, the product from that mismatch incorporates the actual primer, so is now no longer a "mismatch", it's a second (entirely valid) amplicon.
If it's shorter (and it will almost always be shorter, because the best source of mismatch binding is not your starting DNA, it's the other amplicon) then it will also amplify more readily, because shorter templates take less time to copy, and will thus tend to be amplified at higher efficiency per cycle. This is why the second time round the lower band was even more intense (plus the primers that incorporate blunt ends likely are less efficient than the first round primers, which further weakens the gap between match and mismatch -this is why repeating this using those primers on the original template gave you the shorter amplicon only).
BUT: you have a product. And it's the right size. I can see it in your picture.
Cut it out, clean it up, use it.
The beauty of DNA fragments is we can separate them by size, so...do that.
I think that John Hildyard is right but there may be another possibility that is worth checking. If the dna template has 2 complementary regions of sequence so that the template forms a loop in the single stranded state then the enzyme may be reading across the end of the loop producing a short amplimer which then amplifies better as John describes. If this is happening it may be worth adding 6-8% dmso to the pcr to try to stop the secondary structure from forming giving the long amplimer a better chance of amplifying. DMSO might even help if John's scenario is correct because it ensures that only the best primer to template annealinh situation occurs. Try a dmso gradient up to 8% in your pcr ideally.
Thank you very much for your answers John Hildyard Paul Rutland . Yes, next step i will take is to cut (again) my band with blunt ends and sequence it. Anyway, I ordered new primers sacrificing some aminoacids from my protein with no functional consequences.
Paul Rutland I tried with 5%DMSO, but I obtained the same result. I will try a gradient up to 8% just to confirm. Thanks a lot.
It looks like John is correct then. Sequencing might be messy in one direction if the primer can anneal in 2 positions. It would be interesting to hear what the answer is eventually Raúl Villanueva-Romero
Hi,
As per my understanding your one of primer in addition to bind at the end also finding some sequence homology in between the target site i.e. 600 bp from either one of end. That why you still getting 600bp band in gel purified product.
Hope of you understand. I would suggesting you to change the sequences of your primer one by one and verify the said scenario.
Hi everyone,
I checked with another new primer pair and I obtained the same result. I tried to perform the PCR combining them too. Same band at 600bp. Is it possible to get some secondary structures during amplification?
If your product has formed a loop then the enzyme may read across the loop. Sequencing the short fragment will help with an explanation. Try a dmso gradient at the highest annealing temperature that works to try to break secondary structure problems also minimise CG pairing with a pcr with 1M betaine in the pcr mix.. Make sure that you have a no template control in your pcr in case you are getting shortmer contamination in the pcr
It would be very interesting to know the result of a pcr of 4 tubes all of which have No dna added. Use all of the primer pairs outerf-outerR, innerf to inner R, innerf-outerR and outerF-innerR. I am wondering if the original cDNA had an alternate transcript and if your new primers are inside the original primers then if you have contamination of the smaller transcript then all amplifications will reflect this
Hello everyone,
I performed all negative controls and no band was found. I have also sequenced the 600 bp band and it seems to be a jump from exon 4 to exon 13 (last one). I have searched for a splice variant like that but there are none described. I have just found a similarity between the end of exon 4 and exon 12. The sequence of the PCR product at this point is TGGATGAG (Exon 4 end) GTGCAGGCGG (Exon 13 start), while the exon 12 end is TGGTGAG. Although there are just a few similar nucleotides, could it be an explanation?
I have decided to amplify the sequence with 2 amplicons using internal primers. I will clone it using Gibson technology, so i can apply it to unify my whole sequence from those amplicons.
Well done Raúl Villanueva-Romero I am delighted that all your negative controls are negative and that sequencing does solve the problem . If the sequence lengths match that expected then it looks like a 7 base similarity may be enough to produce your effect if that motif exists within the forward primer
i think you should check for other complementary sites for binding of primers within your desire sequence. You need to redesign your primers
Just as a suggestion as I see this has been resolved, an in silico PCR may have been helpful here to obtain amplicon sequences which you could BLAST.
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