joining PCR fragments into a single product

Sergio Alonso sergioal at bbm1.ucm.es
Tue Oct 2 05:49:33 EST 2001


I've joined some PCR products which share a 20bp overlaping ends, just mixing them after
purification and performing 2-3 cycles of PCR without primers. Then reamplifying the whole product
using end-annealing primers:

5'_a--------b_3'
3'_A--------B_5'   product 1

5'_b--------c_3'   product 2
3'_B--------C_5'

After 2-3 cycles of PCR there will be some products like:

5'_a--------b-------c_3'
3'_A--------B-------C_5'

Use primers of 'a' and 'C' to amplify the resultant product. Just add the primers at usual
concentration and perform 20-25 more cycles.

Hope this helps..

Alex ha escrito:

> Chris Boyd <cboyd at holyrood.ed.ac.uk> wrote in message news:<9p1lv0$98u$1 at scotsman.ed.ac.uk>...
> > Alex <ayboro at my-deja.com> wrote:
> > : Hi all,
> > : 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.
> > : Do anybody remember the article and can provide me with the reference.
> >
> > It's also worth looking at
> >
> > 1. Wallace, A. J., Wu, C. L. and Elles, R. G. (1999) `Meta-PCR: a
> >    novel method for creating chimeric DNA molecules and increasing the
> >    productivity of mutation scanning techniques.' Genet. Test., 3,
> >    173-183.
> >
> > where an improved megaprimer method is described that allows catenation
> > of 2 or more PCR products.
> >
> > Best wishes,
>
> Thanks to all of you, but the method that I'm looking for is little
> different. In my case the PCR generated halves have artificial ends,
> different from the gene sequence. So the overlapping region looks like
> this:
>                 \__________________/
>                                /================\
> Therefore direct megaprimer approach is not applicable. I'm thinking
> to chew back the mismatching region of the primers, but hope to avoid
> it.
>
> Thanks,
> Alex




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