Check how long your primer annealing and elongation steps are. You might need to increase the time required to allow for the larger primers to anneal. You may also need to increase your elongation time to allow amplification of the larger 350bp amplicon (if your annealing and elongation steps are separate, sometimes these two steps are combined).

possibly the much longer primers are self annealing (hairpin loop) at the lower annealing temperature of the sequencing reaction. Get some short sequencing primers ( desalted only) .they are very cheap and will work. When you only have a small target not an entire genome as in pcr then short primers will usually anneal in the correct position. Pyrosequencing uses only 14mer oligos and works very well.Can we see one of the .abi sequencing files for a failed reaction please...it may help

Thanks Dr. Donna Nile and Dr. Paul Rutland for your kind response.

Here, I attach one of the failed .abi file. Please take a look.

It seems there was no base-calling at all.

Thank you for that Muhammed can you also tell us the template purification method ( gel purification/exo sap or other) and if you have visible primer dimer after the pcr and the approximate length of the template please?

The primers should be 18–30 to ensures their unique sequence anneal and therefore their specificity. As the length of primer increases, the times to find the particular sequence into the human genome decreases.Primer sequences need to be chosen to uniquely select for a region of DNA, avoiding the possibility of mishybridization to a similar sequence nearby.

However, we don’t want the primer to be too long as efficiency of annealing decreases with increased primer length.

1. Primer Length: It is generally accepted that the optimal length of PCR primers is 18-22 bp. This length is long enough for adequate specificity and short enough for primers to bind easily to the template at the annealing temperature.”

Melting temperature is an issue with longer primers. The length and composition of your primer can affect the efficiency of sequencing. Ideally, you want to have a melting temperature between 52℃ to 58℃. You also have to consider hair pinning and complementary regions causing primer dimers. Finally, when sequencing PCR products, you typically lose approximately 20 bases after the primer. The longer the primer, the greater the amount of sequence you lose.

Dr. Paul Rutland-

There was a single band after PCR and no primer dimer. The length of PCR product ~340bp. PCR product purification was performed with qiagen pcr purification kit.

Everything you have said makes me think you have a sequencing primer with a high Tm. If the Tm of your primer is above 60℃, I would run a two-step cycling method with 60℃ annealing (98℃ for 15 sec, 60℃ for 4 min). The best solution is to synthesize a shorter primer.

Thank you for the file and information Mohammed.

The .abi file shows normal loading and running conditions for the sample but has 2 interesting features. The signal to noise ratio is about 3 ( it should be 100 or more) and there are huge dye blobs ( about 20000 rfu when they are usually less than 2000) of unincorporated sequencing mix at the beginning of the sequence.This is typical of lack of primer binding ( or low template but this is less likely but it is possible that your template is not dissociating in the sequencing reaction) but I am not convinced that this is the case. I think that what is causing this is secondary structure of the primer stopping it annealing properly to the template ( or possibly the template itself has secondary structure issues). In either case the solution may be to run the sequencing reaction as hot as possible ( 64c should work ok) and in the presence of 1ul in a 20ul reaction of DMSO