link two DNAs-a better idea needed

Robert Horton horton at biosci.cbs.umn.edu
Wed Feb 21 13:01:38 EST 1996


 On 20 Feb 1996, jay wrote:

 > Dear netters:
 >   We have two pieces of DNA (400 & 800 bp,in either cloned or PCRed form)
 > that need to be linked/ligated. As shown,they overlap over 100 bp, but have
 > no convenient RE site in this overlap region. And one has several wrong bp
 > (*, induced by degenerate primer).
 >      >
 >      ------------------------------------                    <
 >                                 **---------------------------- 
 > 
 >   We failed with an elongation method: two pieces were PCRed out and 
 > purified (for sure); end-blunted with klenow (rt,15'),heat-denatured and
 > cooled,then elongated with klenow (rt,2hrs),twice; and reamplified with
 > two primer > & <; hoping a hybrid would form as above and the upper strand
 > would be elongated and amplified.
 >   Has anyone done a similar thing and become good at it? or you have a 
 > better idea? Please point a way. Thanks in advance.
 > 
 > jay liu

This looks like a job for Overlap Extension. See:

Horton, R.M., Ho, S.N., Pullen, J.K., Hunt, H.D., Cai, Z. and Pease,
L.R. "Gene splicing by overlap extension". Meth. Enzymol. 217:270-9,
1993.

Sarkar G.  Sommer SS. The "megaprimer" method of site-directed
mutagenesis. Biotechniques.  8(4):404-7, 1990 Apr.

Barik S.  Galinski MS. "Megaprimer" method of PCR: increased template
concentration improves yield. Biotechniques.  10(4):489-90, 1991 Apr.



---
Robert M. Horton
http://134.84.47.3                                       Have a :) day

"Scotty, try flushing the radioactive waste into the ventilation system!"



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