I am hoping users have some suggestions to assist with a problem I am having
with overlap extension PCR/SOE (splicing by overlap extension) and multimer
I can successfully generate a single 1500 bp product (AB) from my first PCR
reaction using a primer (A) just 5' of the area of interest and an internal
primer (B). I can generate a second, 1000 bp product (CD) from another PCR
reaction using a primer 3' of the area of interest (D) and an internal
primer (C). I gel-purify the 1500 and 100 bp products individually.
By design the products AB and CD overlap by ~50 bp, so I have been trying to
generate the overlap extension product AD (2500 bp) by PCR using templates
AB and CD in the presence of primers A and D. The overlap product does not
form (in fact no product is formed).
I have done significant troubleshooting without any success:
-primers A and D will amplify a similar sized fragment (2500 bp) that
originates from a single template (no overlap)
-all PCR stock reagents work in other reactions
-denaturing temperature increased, all the way to 99 deg C, to help melt any
-annealling temperature decreased, all the way to 45 deg C, to promote
-hot start and withhold primers for 1st three rounds over thermocycling to
give overlap a chance to form and be extended before amplification
-addition of DMSO (2% and 5%) to eliminate any secondary structure
I then went back to check the AB and CD templates generated from PCR
reactions 1 and 2, which originally ran at 1500 and 1000 bp, respectively,
and were gel purified. The AB DNA still had a band at 1500, but now also
has additional (lighter or less intense) larger bands--at 3000 and 4500 bp.
To me this suggests multimer formation (concatamers) but I don't know how or
why they are forming (AB product was stored at -20 deg, no heating/cooling,
no ligase). The sequence of the AB PCR product should be:
5'-ttt aCC ATG gct aag aaa acg agc tct aag ggg aag ctg ccT CCG Gga . . .
. . . (~1500 bp) . . .
aCC ATG agc ttc ctg ccc cgc cat cac cac cat tga taT CCG Gct gag gca gcc-3'
In capital letters I tried to highlight above two short (5 base pair)
regions that are identical in the 5' and the 3' ends of the fragment (CCATG
and TCCGG). So, multimers could theoretically form, but it seems unlikely
under any conditions, not to mention while incubating at -20 deg or sitting
at room temperature.
And even if multimers are forming, the majority of the AB template is the
correct 1500 bp size and I don't understand why overlap and extension
wouldn't occur. Perhaps the multimer issue a red herring and there is some
other issue I haven't thought of?