joining PCR fragments into a single product

Sulakshana Mukherjee mukherji at tifr.res.in
Fri Sep 28 12:17:49 EST 2001


Hello!
If you need the ref. for the megaprimer method in 'Biotechniques' then one
of the ref. for this method is :
   Biotechniques 22:438-442 (March 1997)

Incase you need the first paper published on this method then you can get
it from the back ref.s of this paper. I exactly donot remember the
ref. but I think it was published in 1990 in biotechniqes and the author
was Sarkar.

with best regards,
Sulakshana

On 27 Sep 2001 philipp.pagel at cmp.yale.edu wrote:

> > I'm wondering if anybody can help me with a reference. I remember an
> > article (I think at was in Biotechniques) on a simple polymerase based
> > method that allows to generate a single fragment from two PCR
> > generated overlapping halfs. Combining products of 5' and 5' RACE into
> > one contiguous DNA fragment without restriction/ligation has been used
> > as an example.
> 
> It is called "megaprimer pcr". It's very simple: Basically you just dump the
> purified products of both inital pcr reactions into one tube and add the outer
> primers. A few rounds of amplification and you have your merged product.  I'll
> try to draw it here:
> 
>              primer1                     overlap
>              ----->
> product 1    ===================================
> product 2                                ===================================
>                                                                       <-----
>                                                                       primer2
> 
> 
> Now you start the pcr reaction.
> In the annealing step some of the strands of product 1 and 2 will anneal to
> each other and thus function as "megaprimers". That way you get full length
> strands. Now the outer primers can use those as a template for amplification.
> 
> You don't need to run too many cyles since you don't really want to amplify a 
> great deal - all you care for is fully extending the existing megaprimers.
> So it will even work without the outer primers - they just make it more efficient.
> 
> Afterwards you just gel-purify the final product and that's it...
> 
> I don't have a paper but there's a little chapter on pcr mutagenesis that 
> mentiones the technique in:
> 
> Innis, Gelfand, Sninsky
> PCR Strategies
> Academic press, 1995
> 
> 
> cu
> 	Philipp
> 
> -- 
> Dr. Philipp Pagel
> Department of Cellular and Molecular Physiology        phone: (203) 785-6835	
> Yale University                                        fax:   (203) 785-4951
> New Haven, CT 06520, USA
> 

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