Exon skipped during PCR?

Thumb default avatar
Feb 19, 2017


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?



Thumb default avatar
relaxin 11 months ago

Did you use a cloned cDNA as template for the PCR? How big is the exon deleted?

Thumb default avatar
lottiett 11 months ago

Sorry for the late reply, I was not notified of a response. The cDNA was synthesised from RNA extracted from human cells and the exon is 114bp. I have since repeated this experiment and 8/13 of my clones had the deleted exon again; however, this deletion cannot be detected in sequencing data of the cDNA.

Thumb default avatar
relaxin 11 months ago

Since you are using cDNA synthesized from RNA, your template may contain both the full length cDNA and the truncated splicing variant. The truncated variant is normally favored in PCR.

You need to streak your clones for single colony before checking by PCR.