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
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