<x-charset iso-8859-7>Dear Users and Victor
Thank You these very helpful answers that I would like to share with
everybody
Hi Alain
1; It is possible for the Taqman to have 5' Tamara and 3' Fam. From a
historical perspective, it was easier and cheaper to make it 5' FAM and 3'
TAMRA and it still is.
2.Actually the cleavage by Taq is one to two bases from the FAM blocked
5'end. So in a sense the 5'-3' activity is really not an exonuclease
activity but more of an endonuclease.
Hope that helps
Alain:
>
>Could someone help me :
>
>1/ Why a Taqman probe is always 5' FAM, HEX or TET and 3' TAMRA (or any
>quencher) and not the opposite ? (3' FAM, HEX or TET and 5' TAMRA )
>
The TAMRA does not come as a phosphoramidite, I think.
In principle, you should be able to put the quencher at the 5' end. The
BHQ series have the reagents to do that either at 5' or 3'.
>2/ How the 5'>3' exonuclease activity of the polymerase is possible
>since the 5'OH end of the probe is blocked because of 5' (FAM, HEX or
>TET)
It is not an exo activity, it is the 5' flappy endonuclease activity. An
eubacterial DNA polymerase can cut at the hinge of a strand-displacement
reaction, even when the displaced part in say 15 nucleotides long.
THE 5' END IS FAM, HEX OR TET BECAUSE THE MACHINE CAN PUT THOSE ON
AUTOMATICALY. THE 3' END IS TAMRA BECAUSE IT'S ONE OF THE ONLY DYES THAT
CAN BE PUT ON AFTER THE SYNTHESIS. TAQ MAN PROBES REQUIRE A LOT OF HANDS
ON
WORK. I ALSO BELIEVE THAT THERE IS ALSO THE OVERLAPPING ABSORBANCE OF THE
DYES THAT NEEDS TO BE CONSIDERED.
HOPE THIS HELPS SOME.
have a nice WE
ALain
Alpha DNA wrote:
> Bonjour Alain,
> (cette fois la reponse sera en anglais)
>
> 1/ Why a Taqman probe is always 5' FAM, HEX or TET and 3' TAMRA (or any
> quencher) and not the opposite ? (3' FAM, HEX or TET and 5' TAMRA )
>
> It may be mostly by convenience, tradition, and established procedure,
> nobody wants to order expensive dual label oligos, only to hear from
> reviewers of manuscripts and grant proposals that the original method
> must be followed, or that a comparison should be made with the "golden
> standard" of 5'-dye, 3'-quencher). (In a similar line of thoughts, you
> do not want to fix something that is not broken). This being said, we
> did synthesize quite a few oligos of the type "5'-TAMRA,
> 3'-ROX/FAM/HEX/TET", but I am not aware whether the customers used them
> in TaqMan studies. There seems to be a better signal generated when the
> dye and the quencher are at the opposite ends of the oligo, as compared
> to one of them being an internal modification (Livak, K.J., et al.
> Oligonucleotides with fluorescent dyes at opposite ends provide a
> quenched probe system useful for detecting PCR product and nucleic acid
> hybridization. PCR Methods Appl. 1995. 4, 357-362.). In general, probes
> with the quencher dye attached to the 3'-end nucleotide exhibited a
> stronger signal in the 5' nuclease PCR assay than the internally labeled
> probes.
>
> Here is one possibility as to why the 5'-end dye and 3'-quencher may be
> a better architecture:
> Following the displacement step, the 5' to 3' exonuclease activity of
> the Taq polymerase will always cleave first the 5'-end fluorescent,
> signal-emitting dye as a small molecule, free from DNA or with just one
> or two nucleotides attached. In contrast, the 3'-end label will
> inevitably be linked to a larger piece of DNA, because when the nuclease
> degradation advances from the 5'-end, and the remaining (still annealed)
> part of the oligo with the 3'-end label becomes shorter, at the point of
> Tm for that piece, the remaining part of the oligo, including the 3'-end
> modification, will detach from the template, and will escape further
> degradation by the Taq. Therefore, with each consecutive cycle of the
> PCR you will end up having more cleaved 5'-end dye, unattached to DNA,
> or attached to one or two nucleotides only, while the 3'-end dye will be
> attached to a larger DNA cleavage product. At the end, after the last
> cycle of the PCR, as well as in the middle of the PCR, some of these
> 3'-end dyes may anneal back to the template or to other pieces of
> oligos, and may become quenched, while the 5'-end cleaved dye will
> always be short enough to stay non-annealed and emits its signal.
>
> 2/ How the 5'>3' exonuclease activity of the polymerase is possible
> since the 5'OH end of the probe is blocked because of 5' (FAM, HEX or
> TET)
>
> When both dyes are attached to the probe, reporter dye emission is
> quenched. During each extension cycle, the probe is first displaced at
> the 5' end by the Taq DNA polymerase, and then the latter cleaves the
> reporter dye from the probe via its 5'-3' exonuclease activity. Please
> note that this is NOT a cleavage of the 5'-end phosphate or hydroxyl
> group from the last (5'-end) normal or modified nucleotide (or label).
> Your comment that the 5'-end of the TaqMan oligo is blocked means that
> the 5'-end of the first regular nucleotide is used for the
> phosphodiester bond between that nucleotide and the dye, while the dye
> itself does not have a free 5'-end OH (or PO4) group, but that is not
> relevant to the exonuclease. What is attacked by the 5'-3' exonuclease
> activity is the phosphodiester bond between whatever is attached to the
> 5'-end of the penultimate nucleotide, and the penultimate nucleotide.
> Such a cleavage is the normal procedure, regardless of whether you have
> a 5'-end regular (G, A, T or C) nucleotide, or a 5'-end fluorescent dye.
>
> In other words,
> X-A-...
> is blocked, because X does not have a free 5'-end OH or PO4 group (no OH
> or PO4 at the left side of the X), but this blockage does not have
> bearing on the 5'-3' nuclease activity, because the nuclease will attack
> the bond between the X and the A (at the right side of the X).
> The process has two pre-requisitions: a) the Taq polymerase cannot
> cleave the 5'-end dye (or a normal 5'-end nucleotide) from a
> single-stranded DNA, it has to be sitting on a double-stranded DNA,
> building the complementary strand, and reaching the 5'-end of the
> reporter oligo; b) the 5'-end dye or nucleotide must be displaced. In
> fact, the TaqMan probe may be easier cleaved than a normal annealed
> oligo, because its 5'-end reporter dye (and its linker arm) are already
> not annealed ("auto-displaced").
>
> Regards,
> victor
> www.alphadna.com
>
> -----Original Message-----
> From: Association of Biomolecular Resource Facilities
> [mailto:abrf-request@aecom.yu.edu] On Behalf Of Alain LAURENT
> Sent: May 31, 2001 1:22 PM
> To: Recipients of ABRF List
> Subject: Taqman probes
>
> Hello
>
> Could someone help me :
>
> 1/ Why a Taqman probe is always 5' FAM, HEX or TET and 3' TAMRA (or any
> quencher) and not the opposite ? (3' FAM, HEX or TET and 5' TAMRA )
>
> 2/ How the 5'>3' exonuclease activity of the polymerase is possible
> since the 5'OH end of the probe is blocked because of 5' (FAM, HEX or
> TET)
>
> Thank You very much for any answers
>
> BR
>
> Alain
>
> **************************************
> Alain LAURENT Ph. D
> Lab. Manager
> DNA&Peptide Chemistry
> ESGS Groupe CYBERGENE
> Allee christophe Colomb
> 91 035 EVRY
> FRANCE
> Tel: +33 (01 60 87 82 00
> Fax: +33 (0)1 60 87 82 01
> e-mail: a.laurent@eurosequence.com
> http://www.eurosequence.com
> **************************************
--************************************** Alain LAURENT Ph. D Lab. Manager DNA&Peptide Chemistry ESGS Groupe CYBERGENE Allee christophe Colomb 91 035 EVRY FRANCE Tel: +33 (01 60 87 82 00 Fax: +33 (0)1 60 87 82 01 e-mail: a.laurent@eurosequence.com http://www.eurosequence.com **************************************
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