Help with PCR reaction Phusion Flash - wrong sized fragment

[Peter Ellis]

Dwayne Taliaferro wrote:
> There are three possible scenarios.  They're somewhat exotic and I'm sure
> someone on here will suggest a more pragmatic cause/solution
> 
> 1.  The 2kb fragment is nonspecific.  Not likely since you say the 2kb
> fragment contains 1kb from each end of the target.
> 
> 2.  It is likely you are amplifying a version of the gene with a deletion in
> it.  Possible since it is a smaller amplicon, it would out compete the
> larger product.  You may see the larger product if you increase the number
> of cycles.
> 
> 3. There's significant structure in the ends being amplified such that they
> effectively "looping out" part of the sequence or stopping the polymerase.
> These truncated products have enough overlap to amplify each other to
> produce a 2kb product.  Don't think this is likely.  Sounds crazy.

Not necessarily crazy.  If there's a direct repeat within the amplicon 
you're trying to amplify, then partial products from each end might be 
able to anneal to one another and "delete" the section between the 
repeat units.  The smaller product would then outcompete the larger.

In terms of how to fix it, I think the first priority is to get the full 
sequence of the 2kb band, so you can get a handle on how/why it's being 
generated.  Since you can't get the full sequence using only A (+) and 
(-) primers, you'll need to design further sequencing primers inside 
those to get the sequence of the centre of the 2kb fragment.

In regard to the reactions using primer set B, I don't see how the 2kb 
fragment from the  A (+) / B (-) reaction can be the same as the 2kb 
fragment from the original reaction: you know the latter has both A (+) 
and A (-) binding sites or you wouldn't be able to sequence it. 
Sequencing this other 2kb band might also give you clues about what's 
going on.

The suggestion of excising the two 6kb bands from the A/B reactions and 
then using them in an extension PCR is a good one, and may well let you 
get the full amplicon.

Peter Ellis