Exon skipped during PCR?
I have cloned a full-length gene by PCR of cDNA using Phusion DNA polymerase. This PCR also added restriction enzyme sites either side of the gene so the product could be double digested and ligated into an expression plasmid. Sanger sequencing of this expression plasmid revealed that the second-to-last exon was deleted but was not a splice variant. Importantly, this deletion was not present in the original cDNA. I do not think this deletion was a random error because it was present in two of the clones but is it possible that the Phusion polymerase caused such an error during PCR? Is there any other reasons why an exon would be lost during cloning?