Gene synthesis using PCR

[Dr. Duncan Clark]

Hi Folks,

I am having a problem getting four 50mer overlapping oligos to generate
me a full length product using PCR as below.

Oligo 1                             Oligo 3
----------------->             ------------------>
              <--------------------          <------------------
               Oligo 2                        Oligo 4



Oligos have a 10bp overlap and are not kinased. PCR Enzyme is in theory
non-strand displacing. Cycled with low annealing temp 35C for a few
cycles then increased annealing temp and cycled for further 20 cycles.

Oligos 1 and 2 generate 90bp PCR product, as do 3 and 4.

All 4 together do not give 170bp expected product. All I see is 90bp.
Tried various ratios of oligos 1 and 4 ato 2 and 3 in particular less 2
and 3. Tried mixing product of 1 and 2 with product of 3 and 4 and re-
PCRing using 1 and 4 again with initial low annealing temp. Still no
larger product.

Yet to try.

1. Kinase oligos 2 and 3 and use Thermus ligase in with PCR so extension
product of Oligo 1 will ligate to oligo 3 and 4 to 2.

2. Re-synthesise Oligo 3 in opposite orientation. PCR with 1 and 2, use
aliquot of this to PCR with 1 and 3 then re-PCR aliquot of this with 1
and 4.

3. Anneal all oligos and use T4 DNA pol and T4 ligase to extend.

Any thoughts on where we are going wrong. It seemed such a simple thing
to do!

Duncan

  

-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
TEl/FAX 01252376288
http://www.dnamp.com
http://www.genesys.demon.co.uk