First, I optimized the primers which were designed for specific exons, with different Temp (annealing Temp. of PCR) in gradient PCR with standard human blood DNA. After getting annealing Temp. did the conventional PCR with specified Temp. with standard human blood DNA. I got the band intensity good and I did not get the any non specified bands . After optimization of primers performing the PCR with subject DNA samples and sent to sequencing. They said that All samples showed smear indication of degradation. So what should I do for getting good PCR product for sequencing.
Try to decrease the extension time , or use two-steps PCR protocol
the long extension time at 72 produces nonspecific products
try to use the minimum amount of genomic DNA sample. If you get a single amplification product, clean up the PCR reaction using PCR and gel clean up kit to get purified product suitable for sequencing.
Try to use a high fidelity enzyme. Also you can test increase annealing temperature or increase magnesium concentration. All this change will give you a more specific reaction.
I think that degrading is unlikely in a good pcr mix and it is more likely that either you have run too many cycles and the product is starting to self anneal or that one primer is annealing at a much higher temperature than the other and producing more than one product at the lower annealing temperature. Given that you are amplifying many eons then over amplification seems a more likely explanation. Are you measuring the amount of product that you send for sequencing and is it a small amount of your amplified product?
Are you trying to use one of the same primers that you used for PCR for sequencing? If so, then you need to gel purify your product to eliminate any "primer dimer" products that might be present. Even if you can't see them on a gel, they could be present in sufficient molar quantity to interfere with sequencing reactions. Easier solution is to use an "internal" primer for sequencing that is different than either of the amplification primers. And there's no chance your PCR mix contained UNG is there? It's not likely, but that could be a cause of PCR product degradation. Did you run a gel to look at the subject PCR product samples before you sent them to sequencing?
It always a good idea to use sequencing primers different from PCR primers. Purifying PCR product before sequencing is also recommended. We use ExoSap digestion, a simple and quick method for PCR cleanup for downstream sequencing. The exonuclease I removes leftover primers, while the Shrimp Alkaline Phosphatase removes any remaining dNTPs. It is cheaper to buy Exonuclease and Shrimp Alkaline Phosphatase separately and mix them together before use. However, if you live in USA, Canada and Japan you should buy ExoSAP-IT product. It is covered by patents owned by Affymetrix.
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