Are you using GP5+/6+, correct? It is kind of common to find some nonspecific bands using GP primers. I found some higher bands and I read that it can happen, depending of HPV types. After using DMSO you observed lower bands (about 50bp) which probably mean formation of primer dimer. To solve this problem you need optimize the PCR condition, paying attention in the concentrations of primers, magnesium chloride, nucleotides, ionic strength and temperature of the reaction.

I recommend you to, first of all, check out the amount of DNA you are using (I recommend you standardize to 100-150ng) and try to reduce the primer concentration.

Take a look on the paper below.

J Clin Microbiol. 2000 Jan;38(1):357-61.

Improved amplification of genital human papillomaviruses.

Gravitt PE

Are you using GP5+/6+, correct? It is kind of common to find some nonspecific bands using GP primers. I found some higher bands and I read that it can happen, depending of HPV types. After using DMSO you observed lower bands (about 50bp) which probably mean formation of primer dimer. To solve this problem you need optimize the PCR condition, paying attention in the concentrations of primers, magnesium chloride, nucleotides, ionic strength and temperature of the reaction.

I recommend you to, first of all, check out the amount of DNA you are using (I recommend you standardize to 100-150ng) and try to reduce the primer concentration.

Take a look on the paper below.

J Clin Microbiol. 2000 Jan;38(1):357-61.

Improved amplification of genital human papillomaviruses.

Gravitt PE

Hello Priyanka,

You have mentioned that you have used DMSO, but you haven't mentioned about the concentration of DMSO and to what final volume of your reaction. It would be better if you can point out your problem in more precise manner.

Can you tell us the recipe of your reaction mixture, thermal profile you are using machine you are using?

Usually, higher concentration of primer creates the problem. Second is the contamination. I would advise you to look for contamination. If the band is lower than 30, it might surely be primer-dimer. You can clearly identify by running your PCR product in gel-electrophoresis. I think, before you change the magnesium concentration, first try changing the primer concentration because usually magnesium concentration is okay. If you are using DMSO at the same time, then lowering the magnesium will do more harm than good.

Make sure that using too much DMSO will shortens the half-life of your Taq polymerase -- so, be careful with that as well.

Good luck!

In my opinion nonspecific bands is because of your primers are nonspecific and they hybridaze with the genome of the tissue. Low MW are due to primer dimer.

The easiest way is to redesign a new primers with a high rating among them and with your specific target. You'll spend less money and time designing new good ones than doing tests.

In the Literature you can find help and some primers designed by other researchers

Which primers you are using?

May I know which HPV types are you analying? You can try nested polymerase chain reaction to detect specific HPV type.

First of all, I suggest to blast your primers to be sure that you are not amplifying any sequence of the tissue genome you are studying. If you are sure your primers are specific, try to optimize their concentration and the amount of DNA you are using. In my experience, the optimization of these two variables are very important to decrese the inespecificity. Moreover, you can do a gradient PCR to determine the optimun annealing temperature

Good luck!

These noon-specific fragments are sometime call star-effect. Try to reduce polymerase and/or primers concentration if you have blast the primer.

I don't see "Hot-start" in any of these answers. Non specific annealing and extension often occur at low temperature, while setting up the reaction and setting up the tubes in the PCR machine. Often it solves the problem just leaving out the oligonucleotides or the polymerase and adding them when the right annealing temperature has been obtained. This used to be easy when oil was used to prevent evaporation, as these components were added on top of the oil and they incorporated (fell in through the oil) after the heat reached 90oC. Good luck.

Use "Hot-start" DNA polymerase.

Priming starting during set up: Use an incremental activation, to activate the enzyme in steps. For example, 2 minute incubations at 95°C for the first 7–8 cycles.

Annealing temperature not optimal: Run a temperature gradient in 2°C increments MgCl2 concentration not optimal Run a MgCl2 gradient of 0.5mM increments between 1.5 and 4.0mM

Primers not specific: Blast primers to check specificity. Redesign primers.

Overabundance of primer: Decrease primer concentration .

Overabundance of template: Decrease template concentration.

Annealing time too long: Decrease time of annealing step.

Contamination: Check no template control (NTC) for bands.

I agree with the above suggestions. In my opinion, I think readjust the PCR condition (especially optimize the annealing temp.) using hot-start polymerase will be the easiest way to solve your problem.

Tm increased

adding to hot start i am recommend gradient PCR for annealing of the primers at different temperature and choose the specific one, if not better to design different sets of Primers and try again.

There are so many reasons as per my experince you increase annealling temperature, how you designed primers? check primers and its GC content, purify your samples, ckeck out also the primer concentration. Check out all solutions that you are using. Also see dNTP concentration.

Why don't you make the assay easier just buy a RT-PCR kit for HPV? It is cheaper than do-it-yourself.

Wei-Zeng Zhang : can u give me more infromation on RT-PCR kit for HPV detection

www.liferiver.com.cn; email: [email protected] Ask Patty for details. If you mention me to her, you will get a good deal.

Why would you need an RT-PCR kit for HPV whilst is a DNA virus?

Not only in your case, it is common to see the the non specific bands in PCR. My point is if you are getting targeted specific bands with good amplification. Leave non specific . If you are developing PCR diagnostic kits than you follow the suggestions made by Sergio amaro filho.

What exactly is your positive control?

And do you have any -ve control?

Doing a gradient to improve on annealing specificity is good idea. Decreasing the annealing time and increasing the melting time might help as well. Also, primer concetration should not be too high, try to stay below 10nmol. Controls, both positive and negative, are essential. Have you tried to sequence the product found in your negative control?

Mpho Seleka: Why cann't use a RT-PCR kit for HPV? It becomes popular in clinic that a RT-PCR kit for a DNA based assay. Should you need more information, please let me know.

Non-specific amplification of DNA during PCR may be attributed to lots of parameter, as people had mentioned in their previous answer, how ever if you try to amplify the DNA at gradient temperature,or by touch down PCR , it may solve your problem, and all component should also be added in optimum concentration. One more possibility can be contamination of your DNA sample , therefore please check it also.

You should have to re-design PCR primers.

Wei-Zeng Zhang: Thanks a lot for information, I have received details from liferiver regarding kit.

1. Re-do the reaction with a negative control (no template). The non-specific bands could be from contamination of one of your stocks with foreign DNA (probably yours!). If this is a problem, use new stocks, always use autoclaved PCR vials and wear gloves and a lab coat.

2. Increase the annealling temperature. Better yet, use a gradient PCR machine.

3. Redesign the primers and make the 3′ longer. The extra bands may be from similar sequences to your target. Increasing the primer length will make them more specific for your target.

4. Increase annealling time if the non-specific products are shorter than your target. If they are longer than you target, reduce the annealling time.

5. Use less DNA template

I aggree with @Shaima. Performing a gradient PCR should be the first thing to do for a better annealing temperature.

Run a temperature gradient, mayebe your annealing is low. Decrease the amounts of template. Mgcl2 gradient is so useful.