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Overlap extension PCR

Overlap extension PCR - Protocols and Methods Forum

Overlap extension PCR - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 09-08-2004, 10:22 PM
Emily Scott
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Default Overlap extension PCR



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
formation.

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
secondary structure
-annealling temperature decreased, all the way to 45 deg C, to promote
overlap
-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?

Thanks,
Emily






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  #2  
Old 09-09-2004, 01:18 PM
Mark
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Default Overlap extension PCR

Emily Scott wrote:

I have done this once, and met with success so have not had the
need to do loads of trouble shooting. However, I did come across
an article that may help. You may have tried this already, but if
not, check out the use of asymmetric PCR in SOE-PCR constructions.

Gene. 1997 Feb 20;186(1):29-35
"Splicing by overlap extension by PCR using asymmetric
amplification: an improved technique for the generation of hybrid
proteins of immunological interest."
Warrens AN, Jones MD, Lechler RI.

Good luck.

Mark

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  #3  
Old 09-09-2004, 02:29 PM
Michael L. Sullivan
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Default Overlap extension PCR

One idea. Make sure when you do your gel purification that you are
not overexposing the DNA to UV. Some light boxes can be quite strong
and render fragments unusable in subsequent procedures.

If your first set of amplifications are going well and yielding lots
of product, you might not need to gel purify. Using a column based
clean up to get rid of primers might be all you need to do.

Mike



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  #4  
Old 09-09-2004, 05:57 PM
Louis Hom
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Default Overlap extension PCR


FWIW, when I have trouble with overlap extension, I try using super-high
concentrations of the two fragments, either by purifying them together on
the same Qiagen column or by EtOH ppn. So, I typically end up with
probably 500-1000 ng of each fragment in a 50 ul PCR reaction (no
primers). I usually cycle for 10 or 20 rounds, then take 2-5 ul of that
reaction and use it as template in a fresh reaction (1 or 2 x 50 ul) with
the outside primers. It may seem like a black box or handwaving, but it
usually works if I'm having trouble otherwise.
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  #5  
Old 09-13-2004, 10:28 AM
Stein Tore Solem
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Default Overlap extension PCR

In article <[Only registered users see links. ]>, [Only registered users see links. ] (Emily Scott)
wrote:




Dear Emily

This might work (It does for me..): Use 50 ng each of the gel-purified
templates. Cycle at least 8-10x, then add your outer primers (15 pmol)
and do 25-30 additional cycles. To avoid frameshifts and/or
pointmutations in the junctions of the two fragments, I believe it is
important to us a proofreading polymerase in all appplications, but in
particular during the first PCR when the SOE-templates are generated.


Hope this helps,

Stein Tore
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  #6  
Old 02-02-2009, 09:04 AM
Pipette Filler
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Default Re: Overlap extension PCR

Hi all,
I have a typical problem while doing mutagenesis by overlap extension PCR method. While performing overlap extension PCr reaction I am getting many non-specific bands and when I cut my band of interest (as t seems to be) from there and try to amplify it by end primers I am getting a predominant band which is much shorter and not my band of interest. What is wrong with me?

Syam
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  #7  
Old 03-22-2009, 12:42 AM
Pipette Filler
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Default Re: Overlap extension PCR

Hi,

I am having a different problem with overlap extension pcr and seeking assistance from experts.

I can get the band with the expected size, but the band is buried in (or covered with) a very bright lane right from the well (smear). I tried different polymerase and also played with pcr conditions including increasing annealing temperature, shortening time for extension, but I still see a bright whole lane.

Any suggestions are appreciated!

Mary
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  #8  
Old 03-16-2011, 10:36 AM
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Default Re: Overlap extension PCR

I am getting only a smear as well. As far as i could figure out it might be because my initial Tm of annealing the fragments was way to low. Ill let you know what happens. I know the overlap Tm should be higher than whole length to make the process easier.

Last edited by Meserlion; 03-17-2011 at 03:56 PM.
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