Hi René,

Here are some things that I can think of that might help you to determine where your problem lies:

Check for Secondary Structure:

One of the most important things to consider when you are amplifying from a ssDNA template is secondary structure formation, particularly if the GC content is high. Strong secondary structure formation will prevent your primers accessing the template which will prevent that template from being copied by the polymerase in that cycle.

Input your sequence into MFold (linked below) using the Mg2+ concentration of your mastermix (if known) and set the temperature parameter to your annealing temperature, then repeat for your denaturation temperature.

Check if there are any strong stem-loops in your primer binding areas, particularly at your denaturation temperature. If there are, you can increase your denaturation or annealing temperature to minimise this. (Don't worry about thermal degredation of your template, it is well protected in PCR buffer, see second link).

Check your 5' phosphorylated primers:

Check that only one of your primers has a 5' phosphate, because if they both have a 5' phosphate you will degrade everything (personal experience).

Check your digestion:

Lambda Exonuclease has some dsDNA endonuclease activity, so maybe you are over-digesting your template. Try doing a time series of digestion duration.

Check your purification:

Purify some of the dsDNA bands as well and try to amplify those, you might just be having a really low yield in your purification.

If I think of anything else I will add it in. Let me know if I have made an incorrect assumption about your procedure and I will try to fix it.

Regards,

Jacob.

http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form

https://www.neb.com/products/m0262-lambda-exonuclease

Article Effect of sustained elevated temperature prior to amplificat...

Dear Jacob, thank you very much for your comprehensive reply. And thank you for the links. I had a quick look at them.

As far as the secondary structures are concerned, I have never checked them. But, I was able to sequence ssDNA from the same PCR but a different SSCP gel, before. Also the PCR amplifies a variety of amplicons with different sequences. Thus, I think it unlikely that the secondary structures are really the problem here (at least some should work). I will have a look at the folding analysis, though.

Concerning the primers: well, I have separated the ssDNA fragments on the SSCP gel and silver stained the gel (Bassam et al., 1991). I can see nice fingerprints similar to the ones of previous gels. I was also able to amplify and sequence very weak bands before. Thus, I reckon to have amplifieable yields of ssDNA.

Concerning the digestion and purification: Same as above. I can see products on the gel. I just can not reamplify them via PCR for Sanger sequencing. Maybe the DNA was somehow degraded or destroyed during the staining process or while cutting the bands out of the gel. Or I can't elute it out of the gel matrix with the protocol we are using, which is mainly putting the bands into elution buffer and heating it up for 10 min before reamplification PCR.

I am happy to hear more suggestions. ;)

Could be a result of prolonged UV exposure. Did you take longer than normal to excise the bands from the gel, or use a different transilluminator?

Well, if I used UV light, that would be an option. We silver stain the gels and excise the bands on an illuminating table. I assume I have to repeat the gels thouroughly or go for NGS. I can still use the fingerprints to compare the samples. However, thank you very much for your advice, Jacob!