blocking a dna adapter / linker against 5->3 fill in ?

Ralf Sigmund ralf.sigmund at mpihan.mpg.de
Mon Sep 6 07:47:26 EST 1999


Thanks to all of You for Your Replies.
Wheras Gibco told me that they would non offer dd-nucleotides in the
3'-terminal position,
MWG Biotech does offer them.
The MWG Biotech customer support person did even tell me about the
possibilty to use an amine group.
Amine is cheaper than didesoxy, because it is needed more frequently.
I finally ordered today from MWG, because I hope they will be able to achive
a higher product purity in comparison to the use of terminal transferase.

It is a mess that Newsgroups do not support formatted text or graphics...
This makes it quite hard to explain things...

What i intend to do is:

5'-  TCGTTACGAAGCACTTATAACGATGAGTCCTGAG-3'
3'-
                                                        ddTACTCAGGACTCGC -5'

      on the right side there should be a GC overhang in the lower oligo.
the terminal C of the lower oligo is Phosphorylated.
      ligation should yield:

5'-  TCGTTACGAAGCACTTATAACGATGAGTCCTGAGcgnnnnnnnnnnnnnnnnnnnnnn-3'
3'-
ddTACTCAGGACTCGCnnnnnnnnnnnnnnnnnnnnn -5'

later I want to do PCR with the Primer

5'-  TCGTTACGAAGCACTTATAACG-3'

which is exactly complementary to the void space in the lower part of my
adapter.
so this pcr should never work, because the primer has nothing to bind to.

If I use two different adapters in my ligation - one like the one described
above and one normal without the didesoxy, than I can amke shure, that
fragments which got ligated to didesoxy-blocked adapter on both sides will
not be amplified in a subsequent pcr

 This does not help against ligation of the normal adapter on both sides.

However the pcr-primer for the normal adapter does not only bind to the
adapter but also bind to the insert with its three 3'-terminal sequences.
so the first three bases of the unknown insert must exactly match the
primer's three bases by accident.
the probability of this is one in twohundretfivetysix.

If the unblocked adapter is ligated to both ends of the fragment and the
fragments three first bases on both sides to match the used primer than
there would be a pcr product.
as I use only one of the 64 possible primers at a time this means the a pcr
amplified fragment would have to have the same thre bases on both ends.
this case is rare.

so most products will result from the blocked adapter linked to one site and
the unblocked to the other side.
(the first strand made by priming on the unblocked side creates a new full
length strand :

5'-  TCGTTACGAAGCACTTATAACGATGAGTCCTGAGcgnnnnnnnnnnnnnnnnnnnnnn-normal
adapter-3'
3'-    <           <         <
<----------------------synthesis-------------------------------------------x
xx-PRIMER -5'
)

I hope I was able



Chris Boyd schrieb in Nachricht <7qvrnl$k9$1 at scotsman.ed.ac.uk>...
>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,
>--
>Chris Boyd                      | from (but not \  MRC Human Genetics Unit
>Christopher.Boyd at hgu.mrc.ac.uk  | much longer)  /      Crewe Rd, Edinburgh
>http://www.hgu.mrc.ac.uk/Users/Christopher.Boyd          EH4 2XU, SCOTLAND





More information about the Methods mailing list