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I was wondering if anyone has ever seen large deletions (~40-50 bp) occur when using phusion polymerase to clone a gene. I have recently tried using Circular Polymerase Extension cloning to clone a 200 bp insert into a 6.5kb plasmid and obtained two separate clones with 40bp+ deletions in the center of the insert.

Phusion has a 5'->3' exonuclease activity, but I didn't think it could cut in the center of a sequence. While our inserts were created with oligo synthesis, it is my understanding that such large deletions are rare with oligo synthesis. If you have heard of this happening please let me know.

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I don't think that it would be an exonuclease issue. Rather it may be an issue with hairpin formation during amplification. Are you sure you have the complete 200 bp insert? –  bobthejoe Jun 16 '12 at 6:06
    
I've just seen a 12 bp insertion in a cloned sequence amplified with Phusion polymerase. –  user4251 Aug 16 '13 at 20:03

1 Answer 1

In my own personal lab experience, I got some unexpected results using Phusion polymerase. However, I have not seen deletions, only insertions (ranging from single bases to 3 tandem copies of a primer sequence). This is not an answer to your deletion question, but it does suggest unusual things might happen.

I have seen deletions with T4 DNA polymerase during "blunting" reactions. According to the NEB website:

Q10: Is T4 DNA Polymerase active at room temperature?

A10: We suggest 12°C. The DNA ends "breathe" at higher temperatures allowing the exonuclease to remove nucleotides past blunt.

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