optimizing overlap PCR

Maximilian Haeussler via methods%40net.bio.net (by maximilianh from gmail.com)
Fri May 18 11:42:29 EST 2007


Hi experts,

I want to do overlap-extension  (fusion) PCR in a medium-scale
project, learning from articles [1], [2], [3] and [4]. I have to
optimize for simplicity (no purification) and highest possible yield
of the final product in ug of DNA (it is meant for electroporation,
I'll need around 10 ug of DNA in the end).
I'm doing nested primers, outer primers at a TM of 70, overlap primers
with TM60, to amplify only the fusion product in the final PCR (as
recommended in [4], I've drawn a schema of this at the end of the
message). This seems to work now though with many different primers.

There is a sufficiently pure product in the end. I don't know,
however, how to optimize my final PCR for maximum yield. (It seems
that I have to use some sort of PFU, I don't know why, perhaps I need
the exonuclease activity to get rid of non-overlapping part C (see
schema))

What would you recommend:
- Use rather more polymerase or mix taq/Pfu for higher yield?
- Is it worth buying the highly expensive Phusion/... enzymes or would
you rather buy some cheap polymerase for the final PCR?
- For the extension PCR: NEB is selling a special polymerase, Vent
(exo-) for overlap extensions, do you think this is worth the money?
- Apart from increasing polymerase concentration and primers, is there
anything else that can be done for maximum PCR yield (I cannot find a
good reference for "maximum pcr yield")

schema legend:  -- = template, ~~ = primer, \ = non-overlapping DNA,

                                           \   \   C: tm60
                                            \
                                             \
        ----------FRAG1-------     +---------FRAG2-----------------
~~~~~~~             ~~~~-~~~                 ~~           ~~
A tm70:tm60            B  tm60:tm60              D 60       E 70
~~~
F tm70

outline of the PCRs:
Frag1 and Frag2 are to be joined into one.
PCR1 on genomic DNA:  primers A+B (anneal at 55)
PCR2 on plasmid:           primers C+D (anneal at 55)

mix 1ul of PCR1 and PCR2 into new tube, then run
10x at 50 deg. without primers, add primers F and E, cycle 30x at 60
deg.  annealing temp. (don't really know if all of this is necessary)
No gel-purification in the end, as there is only one (visible) band.

Any general experiences or references on overlap extension PCR are
also appreciated,

cheers,
Max

[1] Splicing by overlap extension by PCR using asymmetric
amplification, Gene 1997
[2] Fusion PCR, a One-step variant of the Megaprimer Method of
Mutagenesis, Biotechniques 1998
[3] Construction of long DNA molecules using long PCR-based fusion of
several fragments simultaneously, NAR 2004
[4] Rapid Expression of Functional Genomic Libraries, Journal of
Proteome Res 2006
--
Maximilian Haeussler,
bioinformatics grad student,
CNRS Gif, France


-- 
Maximilian Haeussler,
skype: maximilianhaeussler



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