ICON probe

Outline

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.

 

Product features

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 complex reactions”

 

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 cytosine.



Examples of experiment

1.  ICON Probe induces osmium complex formation only with the target methylcytosine

2.  Selective quantification of methylated DNA with ICON Probe (a model experiment)

3.  Unaffected by other coexistent methylated regions during quantification of methylation

4.  Measurement of the amount of methylation in each mouse genome tissue

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 Price ( Shipping fee is necessary other than product price .)

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 by phosphorylation)

*For the target methylation candidate, one sequence for sense chain and another for antisense chain are needed as the ICON Probe.

♦Sequence design points for ICON Probe (for ICON Probe set)are shown here.

Guaranteed amount: each 5 nmol

Price: ¥78,000

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)

*Precaution

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.

 

Reactions

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 () (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 subsequent experiments

 

Quantification

A portion of the desalted filtrate is combined with the fluid for PCR containing the primer, dNTP, DNA polymerase and SYBR Green I.

 

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.

 

Calibration curve

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 growth curves.

 

Reference information

♦How to prepare samples for calibration curve

1.  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

2.  The samples obtained in Step 1 above are methylated with a commercially available methylation kit.

→ 100% methylated sample

3.  Mixtures at varying ratios of 1 to 2 are prepared, to yield samples of intermediate concentrations.

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