blocking adapter... ==> "extender PCR"

[Roland Hübner]

In article <7qvrnl$k9$1 at scotsman.ed.ac.uk>, chrisb at hgu.mrc.ac.uk (Chris
Boyd) wrote:

> Nicolas von Ahsen (nvahsen at hotmail.com) wrote:
> : On Fri, 03 Sep 1999 12:49:04 GMT, juamber at my-deja.com wrote:
> 
> : >In article <7qjidt$1b0g$1 at gwdu67.gwdg.de>,
> : >  "Ralf Sigmund" <ralf.sigmund at mpihan.mpg.de> wrote:
> : >
> : >> Now I would like to block the 3'-terminal base of the bottom linker,
> : >so that Taq-Polymerase cannot fill in the 5'-3' direction.
> : >> I thought about ordering a oligonucleotide with a 2',3'-didesoxy group
> : >in this position.
> : >
> : >Why not ordering a modified oligo with an amine group in the 3'
> : >position? It also blocks the taq (Siebert et al. 1995. NAR 23:1087-1088)
> : >Don't know about prices.
> : >
> : >Juamber
> 
> Yes, but I imagine this will also block ligation.
> 
> : Or also you may use a 3' phophate group which is easily intruduced by
> : starting DNA synthesis from a phosphate CPG. Four 0.2µM columns cost
> : $60 (GlenRes). Almost every Oligo-supplier should be able to do that.
> 
> This will also block ligation unless the 5' end of the other DNA partner
> in the ligation is dephosphorylated. And even then I don't know if ligase
> will join a 3' phosphate to a 5' CH_2 -- I'd guess not.
> 
> 
> To recap:
> 
>    5'-  GNNNNNNNNNNNNNNNNNNACGATGAGTCCTGAG   -3'
>    3'-                        TACTCAGGACTCGC -5'
> 
> If I understand correctly, you want to make the above ligatable adapter
> in such a way as to prevent Taq filling in the bottom strand (but by
> implication don't care about it filling in the top strand)? It seems to
> me this is impossible: any permanent modification of the bottom oligo
> to stop Taq activity is likely also to screw up ligation, which as far
> as I know requires a 5' phosphate and a 3' OH group (but I would be
> glad to be corrected on this).  My advice would be to take steps to get
> rid of Taq polymerase activity at the appropriate time and make the
> above oligo with unmodified oligos (or 5' phosphorylated oligos if the
> strategy demands them).
> 
> Best wishes,


Hmmmm,

 what about "extender PCR" (recent Biotechniques)? 

 a) ligate non-modified adapter on

 b) make the lower oligo so as to block 3'- with desired ddNTP (Taq; 5 min at 
    68-75oC)
 
 c) now, PCR


 Somebody compared the power of this approach with:

 i/ first round(s) of PCR with "gene-specific" primer 
 
 followed by 

ii/ addition of adapter primer and exponential cycling

 Definitely cheaper, eh?


Kind regards,
 Roland