Hi Forest,

I would try to play around with the PCR conditions and different polymerases. Did you try to add DMSO (e.g. 5%) to your PCR to break secondary structures of DNA? And for example the Phusion polymerase from NEB comes with a "special" buffer for GC-rich sequences (as an alternative for the standard HF PCR buffer). I've had good results with that one.

Cheers,

Tobias

Try to use primer3 (on-line tool) or ,if you can, oligo6 (software).

Hi Forest,

I would try to play around with the PCR conditions and different polymerases. Did you try to add DMSO (e.g. 5%) to your PCR to break secondary structures of DNA? And for example the Phusion polymerase from NEB comes with a "special" buffer for GC-rich sequences (as an alternative for the standard HF PCR buffer). I've had good results with that one.

Cheers,

Tobias

I had similiar problems as well, and unfortunately there is not "the" answer for this problem. Sometimes shifting of primers can help, sometimes nested PCR works better. You could also try to play around with the PCR conditions, e.g. salt concentrations, annealing temperature or trying to run a few cycles at a less stringent annealing temperature and the go higher with a shorter annealing time which fits your template size.

Other things helpful might be the use of DMSO or Betaine.

I agree with Markus, PerlPrimer is a great tool. I use it for all my primer designs. Unfortunately it hasn't been updated for a while now, so automatic sequence import doesn't work.

First, I agree with ePrimer3 software, it gives you lot of advanced thermodynamic choices and is free. I think you should design several (2-3) pairs of primers to have options, nested PCR is not a bad options and has “saved” our work with virus promoters a couple of times. The problem with CG-rich sequences is that is a strong competition to bind primers at alternative sites on the template, thus you end up having a smear or, worse, a sharp non-specific PCR product (it happened to me). Therefore, you will have to work hard to optimize chemical and thermal conditions. In that respect, choose e processive enzyme (ie with proofreading activity), and add some coadjuvant such as DMSO (it really ameliorates the inhibitive effect of competitive binding on the yield function by increasing the melting rates at those sites) or betaine. We usually prepare an enhancer solution 5X (dark 4ºC) with betaine (Sigma B-0300) 2.7 M + DMSO (Sigma D-8418) 6.7% + DTT 6.7 mM. You can try 2x, 1x, 0.5x concentrations in your reactions to see which one work with each primer pair.

7-Deaza-dGTP will work better for highly structured regions, and sometimes is the only option. It also improves your sequencing results (another chapter for problematic DNA templates).

The annealing temperature and time are also important in this type of PCR. Once you reach a “optimum” temperature (or thermal profile ie touchdown), the annealing time must be long enough to form the ternary complexes at the correct template site, but too long annealing time creates the opportunity for ternary complexes to form at incorrect binding sites.

You can also try with this published protocol

http://download.bioon.com.cn/view/upload/month_0905/20090517_c72a692db2666444df2bYy2EadrYtKcX.attach.pdf

Wish you luck!

GC Rich PCR System (Roche) and Phusion HotStart (Finnzymes: I suppose it is the same of that of NEB suggested by Tobias) with DMSO gave me good results.

What do you know about the results you are getting? For example, are the PCR products distinct sizes, consistently smaller or larger? Are you able to get them sequenced, to tell you about the what is being amplified? This may give you clues to why these sequences are being amplified.

Perhaps there is information in these products that could help you. Do you know they are not related to the region you are interested in?

I would not always trust that the database contains the same information as your samples. Are the primer sequences checked against the human database for binding to other sites, or repetitive regions?

Primers matching as little as 6 bases to the DNA is enough to amplify it.

I give you more questions than answers, but I would look in more detail at the results you are getting.

We had similar problems years ago.

We used nested PCR approach and it worked.

Optimising the PCR conditions may help (using for example GC rich amplifying systems), but I would not worry to much getting non-specific fragments as well.

I would clone and sequence some of the bands if there are distinct bands on the gel after nested PCR.

Hi Forest,

It sounds like you've done some extensive testing, but I just wanted to make sure that you tried an annealing gradient with this primer set. I have found that merely shifting the annealing temperature 0.5 - 1'C higher has reduced a lot of non-specific amplicons in my PCR. Annealing gradients tend to be the first step of PCR design, so I apologize if you've already tried it. And, as many others have already stated above, if you can't get it to work with this one primer set, you may have go for nested PCR.

What is you final end-point for this PCR? Is it qPCR, are you sequencing, are you cloning? If you're just cloning or sequencing, you could skip the trouble-shooting and just gel-extract your PCR product of interest.

Good luck!

my experience is that to design longer primers, with 2 steps PCR, saying denature-enlongation PCR cycles, without anealling step. Keep less than 6 GC in the last 10 bases in the 3 ends of the designed primers. Try to avoid GC rich region to match your 3 ends of the primers, by simple making longer primers. 2 step PCR works well with very long primers as soon as the 3 ends not being GC rich. Other suggestions like to choose GC rich compartable polymerase, or add DMSO etc may also just work as well.

Hi.

I use the Oligo Design and Analysis Tools from Integrated DNA Technologies. Here is the link: http://www.idtdna.com/scitools/scitools.aspx

I had good results with those tools. Hope it is useful.

I would add a few more things from my sequencing experience with pre-miRNA regions:

1. make sure you extend your primers to have a high annealing temperature and get rid of the background. Primers of 40-50 nt can help a ton and are not much more expensive. But avoid 4 G stretches in your primer, they form Quadruplexes and can hinder your PCR/primer synthesis. 65-70C depending on the polymerase you use, you can then run it as 2-step. I use for melting temperatures the Weizman Institute Oligo Melting tool, it feed Primer 3 with a specific set of conditions I found works very well in predictions for many years - use Breslauer Enthalpy results with Primer 3, not the other temperatures it offers.

2. Place your primers at least 50 nt upstream/downstream of the start of your structure, better 100 nt. If the polymerase is for sure out of the cycling initial step and has run for a bit your chance of making it over structured regions are much better.

3. I found background gets worse with DMSO but betaine usually works well. Gradient PCRs to figure out the best profile can help a lot too of course.

Hi Froest, somethings it will depends on which Taq you used and you would also have to optimise your MgCl2 concentration. In the old days we could also use DMSO to lower the Tm of primers. But, nowadays ABI ampliTaq Gold kit contains high CG rich PCR buffer. Could give it a go and see if you can get the region you want. You could also consider using Oligo 6 software to redesign your primer. Good luck!

Hi Forest, in the past I had the same problems with the 5 UTR of my gene that I needed to amplify for my constructs. After several attemps, I tried overlap PCR with success. If you design primers in non-conflictive zones (probably you would need to test various primer pairs) that amplify fragments overlapping at least 20 nt, you do the partial PCRs separatelly and then you can amplify the whole fragment using the primers corresponding to the ends of the comlete fragment:

http://en.wikipedia.org/wiki/Overlap_extension_polymerase_chain_reaction

If still you have issues, another option that I also succesfully tried was to amplify with PCR the whole fragment except for the problematic end. You can obtain the template for the problematic end by ordering the 2 complementary fragments for this end, that should overlap with the normally amplified fragment. Then you have to hybridize both complementary strands of the end and finally you do the overlap PCR as I described before. (sorry for my english, if I did not explain myself correctly, you can contact me by email).

It is useful also to use a good high-fidelity Taq polymerase that usually is accompanied by an amplification buffer for difficult amplicons.

Hope it helps! Good luck!

I have had very good results with touch down PCR with genomic DNA. I typically run the first 3 cycles at 6 degrees above the suggested Tm, the next 3 cycles at 3 degrees above suggested Tm and then 30 cycles at suggested Tm. Majority of spurious amplifications are eliminated this way and you build up enough concentrations of correct amplicons in the reaction which drive the reaction in the subsequent 30 cycles. Polymerases have also an influence on how well certain regions are amplified. Between Qiagen's Taq pol with Q-solution and Takara Ex-Taq polymerase, I have been successful at amplifying majority of my genomic DNA PCRs. You can also do an approach similar to RACE where you use only one primer initially to hopefully yield linear amplification of one strand of the region you want to amplify (20-25 cycles), purify the reaction and follow that up with the primer pair for a regular PCR. You can try two separate reactions in initial linear amplification and hopefully one of them will amplify more specific template from the region of interest.

Hi Forest,

You can use Primer3 online server for primer designing. It is free online at web address: frodo.wi.mit.edu/

The non specific bands can be overcome by optimizing the annealing temperature. It is better to use gradient PCR rather then conventional thermocycler. If you have facility for Real time, it is better to use.

Surya