11
$\begingroup$

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.

Improve this question
$\endgroup$
2
  • $\begingroup$ 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? $\endgroup$
    – bobthejoe
    Commented Jun 16, 2012 at 6:06
  • $\begingroup$ I've just seen a 12 bp insertion in a cloned sequence amplified with Phusion polymerase. $\endgroup$
    – user4251
    Commented Aug 16, 2013 at 20:03

1 Answer 1

Reset to default
3
$\begingroup$

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.

Improve this answer
$\endgroup$

You must log in to answer this question.

Not the answer you're looking for? Browse other questions tagged .