Just that: ¿will PCR work if the forward and reverse primers are identical? Assuming of course the template has appropriate binding sites.
In particular, I am looking at two-round PCR, where the first round introduces overhangs which are used by the second round PCR as primer binding sites. This is fairly common (e.g. see Figure 2 in http://rsos.royalsocietypublishing.org/content/2/7/150088), but I am wondering whether it would still work if the overhangs on either end are identical (and so are the primers for the second step PCR).
I would expect it to work.I have run many pcr reactions where one primer is capable of generating a specific (but wrong) product so if you have a short sequence where one primer anneals in the correct orientation at each end then I would expect a product. Sequencing with these primers would be a problem
I believe it might still work.
I use the whole PCR product as primers for PCR based cloning into plasmids, wherein the terminal 30 bp sequences are complementary to the insertion sites of plasmids.
Theoretically both primers are self annealing, because they are PCR products, and it still works as intended.
However, I do use additional steps to confirm if the PCR cloning worked or not.
So in my view you must have a secondary method of confirming the results from the PCR.
Best
Sudeep
If they are identical, then there will be two issues at play. First, you will need to be certain that the sequence is not a palindrome or complementary to itself. If it is, you will get primer dimers that could compete out your reaction. You will also want to check for subsequences that might lead your primers to dimerize.
Second, if they anneal to different loci (as they should if they are not palindromic), you could run into trouble if one site has an architecture with higher affinity for the primer sequence than the other site. This should be rare for most templates, though if you are using a template that is heavily modified or difficult to denature you may run into trouble getting both sites equally accessible.
These two caveats aside, there’s no inherent reason it should not work. It is situationally dependent on the primer design and template. If you are failing due to a dimer, that should be obvious when you run the PCR product on a gel, because you’ll either have a big streak or a really small but bright band that is nowhere near the length of your desired amplicon (unless your amplicon is similar in size to your primer, which would make me question why you’re doing PCR at all).
If it’s failing due to the second reason you will likely see nothing when you run the product on a gel, as you won’t achieve the “C” part of PCR and won’t get exponential amplification. You might see some very faint products of inappropriately long sizes on a gel, but this is unlikely.
Thanks to everyone for your insight. The primers will not be palindromic (in fact I can choose them to be whatever I want, so I can avoid palindromic sequences by design). However, if the forward and reverse primers are identical, the product must necessarily contain the primer binding site in both forward and reverse-complement orientations, so my only remaining concern is whether the individual product strands will tend to form hairpins rather than anneal to the primers in a given cycle. In any case, I now feel more encouraged to give this a try, so thanks again!
It might work, but it might be very messy as you have a lot of chances for non-elongating binding. As you suggested, there is a real possibility your amplicons will form hairpins during annealing. I think your amplicon length can mitigate this somewhat (longer probably reduces slightly the hairpin stability and formation). Conceptually, you are making a molecular beacon. You should use mFold or another folding calculator to see how stable your end product is.
- Badges
- Science method