ICON Probe is a totally new site-specific DNA
methylation detection probe. It can be
used for research on epigenetics related to various biological phenomena and
diseases. As compared to conventional
techniques, this probe enables, for example, quantitative analysis of DNA
methylation at the target site of genome in shorter time and with smaller
amounts of sample, without causing damage to the sample.
Our company has concluded a licensing
agreement with Kyoto University and now serves as the only manufacturer and
distributor of ICON Probe in Japan. It
is possible to create oligonucleotides tailored to client’s desire by using the
modified nucleotides adopted for ICON Probe.
Clients can thus develop new applications tailored to their objectives.
ICON Probe is the name for the site-specific
methylation detection probe developed by Dr. Akimitsu Okamoto (Okamoto
Initiative Research Unit, Frontier Research System RIKEN).
Research topics by Dr. Okamoto are given here.
Linked to: RIKEN
“Rapid identification of the target methylation site through metal
ICON Probe is composed of a DNA sequence
portion (the DNA fragment complementary for the DNA to be checked as to
methylation) and a modified nucleotide portion.
After the target sequence is identified by the DNA sequence portion of
this probe, the modified nucleotide binds via osmium to methylcytosine, to form
a cross-link between DNA chains. This
reaction with non-methylated cytosine is more than 400-fold slower, thus
enabling clear-cut distinction between methylcytosine and non-methylated
Examples of experiment
Probe induces osmium complex formation only with the target methylcytosine
Selective quantification of methylated DNA with ICON Probe (a model
Unaffected by other coexistent methylated regions during quantification
Measurement of the amount of methylation in each mouse genome tissue
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ICON Probe set
(real-time PCR detection system)
Set of sense chain target probe and antisense
chain target probe
(sequence length: 20-mer, 3’-terminal modified
*For the target methylation candidate, one
sequence for sense chain and another for antisense chain are needed as the ICON
♦Sequence design points for ICON Probe (for
ICON Probe set)are shown here.
Guaranteed amount: each 5 nmol
Demands to modifications (change in sequence
length, other various modifications) can be met.
|• Order via E-mail → Download the specific form and send it to the address
for order given below.
♦Specific order form is given here (Excel, PDF)
|E-mail address for order :
Regarding ICON Probe
ICON Probe is manufactured and distributed
under a licensing agreement between Kyoto University and Gene Design Inc.
ICON Probe is a registered trademark of GeneDesign, Inc.
Model experiment protocol
Selective quantification of methylated DNA
sequence with ICON Probe (use of real-time PCR)
The protocol given here pertains to a model
case at early stage. Better settings may
be identified through further evaluation of concentration of each solution,
etc. So, this protocol should be viewed
only as reference information.
Target DNA solution (100 nM) 5 μL
ICON Probe mix (one for sense chain and
another for antisense chain, each 100 nM) 5 μL
Aqueous potassium hexacyanoferrate (Ⅲ) (1M) 5μL
Buffer mix (Tris-HCl (pH = 7.7, 100 mM), EDTA
(1 mM), NaCl (2M)) 25 μL
These elements are mixed in a reaction tube,
followed by 5-minute heating at 95oC and rapid cooling at 0oC
Addition of aqueous potassium osmate (25 m) 10
μL, followed by one-hour heating at 55oC
Desalting, and the filtrate is used for
A portion of the desalted filtrate is combined
with the fluid for PCR containing the primer, dNTP, DNA polymerase and SYBR
Real-time PCR is carried out.
(Conditions: 95oC for 5 seconds →
60oC for 10 seconds → 72oC for 15 seconds) x 50 cycles
Real-time tracing (excitation at 470
nm/detection at 510 nm) is carried out, and the data obtained are analyzed.
The above-mentioned sequence of reactions is
performed also on the standard samples (0% methylated sample, 100% methylated
sample, etc.), to prepare a calibration curve from the ascending point of their
♦How to prepare samples for calibration curve
About 300 bases, containing the target sequence, are amplified by PCR,
followed by purification (the sample in which the methylated portion has become
non-methylated is amplified by PCR).
→ 0% methylated sample
samples obtained in Step 1 above are methylated with a commercially available
→ 100% methylated sample
Mixtures at varying ratios of 1 to 2 are prepared, to yield samples of
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