Exon skipped during PCR?

Non-professional visitors (i.e., lay people, high school and undergraduate students) or professionals from other fields should use this forum for general questions regarding molecular biology. No guarantee that they'll be answered but you can always try!

Moderators: r.rosati, mchlbrmn

Exon skipped during PCR?

Postby lottieTT » Feb 19 2017 12:55 am

Hi,

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?

Thanks,
Lottie
lottieTT
newcomer
newcomer
 
Posts: 2
Joined: Feb 19 2017 12:36 am

Re: Exon skipped during PCR?

Postby relaxin » Feb 21 2017 11:32 am

Did you use a cloned cDNA as template for the PCR? How big is the exon deleted?
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
relaxin
PI of Posters
PI of Posters
 
Posts: 7156
Joined: Jan 11 2006 12:40 pm
Location: Mauna Kea

Re: Exon skipped during PCR?

Postby lottieTT » Apr 19 2017 10:22 pm

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.
lottieTT
newcomer
newcomer
 
Posts: 2
Joined: Feb 19 2017 12:36 am

Re: Exon skipped during PCR?

Postby relaxin » Apr 21 2017 12:04 pm

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.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
relaxin
PI of Posters
PI of Posters
 
Posts: 7156
Joined: Jan 11 2006 12:40 pm
Location: Mauna Kea


Return to Student Questions

Who is online

Users browsing this forum: No registered users and 1 guest