Table of contents. I. Description...2. Kit Components...2. Storage...2. Required reagents and equipment...2. V. Protocol...3. Example Experiment...

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1 PCR FLT3/ITD Mutation Detection Set Cat.# 6632 Table of contents I. Description...2 II. III. IV. Kit Components...2 Storage...2 Required reagents and equipment...2 V. Protocol...3 VI. XII. Example Experiment...4 Reference...6 XIII. Note...6

2 Cat.# 6632 PCR FLT3/ITD Mutation Detection Set I. Description: This kit is used to detect internal tandem duplications (ITDs) which occurs around of the JM (Juxtamembrane) domain of the FMS-like tyrosine kinase 3 (FLT3) gene. It has been reported that ITDs of the FLT3 gene are found in approximately one-third of acute myelogenous leukemia (AML) patients and in approximately 3% of myelodysplastic syndrome (MDS) patients. The mutations are most commonly found in exon 11 of the JM domain, but can be found in intron 11 and exon 12 as well. The primers contained in the kit are designed to allow amplification of the entire region from exon 11 to exon 12, which ensures reliable detection of ITDs. The procedure used, which essentially consists of PCR amplification using genomic DNA obtained from bone marrow fluid or blood and determination of the size of the amplification product using electrophoresis or the Agilent 2100 bioanalyzer, detects ITDs when present in 5% to 10% of the genomic DNA. The kit contains both normal and mutant controls to verify its accuracy. AML patients with ITD mutations tend to have poor prognoses and are often refractory to treatment; molecular target drugs that specifically inhibit the kinase activity of FLT3 are currently under development. The kit can serve as valuable tools in the research and development of this class of drugs. II. Kit Components (for 50 reactions): 1. FLT3/ITD-1 Primer (10 pmol/ μ l)...50 μ l 2. FLT3/ITD-2 Primer (10 pmol/ μ l)...50 μ l 3. Control Template (Mutant) (1 pg / μ l)...10 μ l 4. Control Template (Normal) (1 pg / μ l)...10 μ l III. Storage: (for shipping and storage) IV. Required reagents and equipment (other than this kit) [Reagents] TaKaRa Ex Taq TM (Cat.# RR001A) NuSieve R 3:1 Agarose (Lonza) TAE buffer DNA Marker [100 bp DNA ladder (Cat.# 3407A/B)] Loading buffer (6X : 36% glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, 30 mm EDTA, this loading buffer is supplied with Takara s Takara's DNA Ladder.) Ethidium Bromide. [Instrument] Authorized thermal cyclers Electrophoresis apparatus Power supply for electrophoresis UV transilluminator Polaroid camera for electrophoresis gel [Equipment] 0.2 ml PCR tube [TaKaRa Micro PCR Tube (Cat.# 9047) 0.5 ml, 1.5 ml Microfuge tube Micropipette and tips resistant to aerosol. [For analysis with Agilent 2100 Bioanalyzer] 2100 Bioanalyzer (Agilent Technologies) DNA 1000 LabChip Kit (Agilent Technologies) 2

3 PCR FLT3/ITD Mutation Detection Set Cat.# 6632 V. Protocol: A. Preparation of DNA from bone marrow fluid and blood Prepare the genomic DNA from bone marrow fluid or blood using a standard method such that detailed below. Do not use heparin as an anticoagulant when collecting bone marrow fluid and blood, as heparin inhibits PCR. Use disodium EDTA or a similar reagent instead. Give attention to prevent contamination with erythrocyte components during genomic DNA preparation, because such contamination can inhibit PCR reaction. 1. Centrifuge 1 ml of bone marrow fluid or 5 ml of blood at 1500 rpm for 5 min. 2. Wash the precipitate in 5 ml of Hemolysis Buffer, and centrifuge at 1500 rpm for 10 min. * Hemolysis Buffer: 10 mm Tris-HCl, ph7.6, 5 mm MgCl 2, 10 mm NaCl 3. Repeat the washing step of 2. twice more. 4. Add 1.5 ml of Lysis Buffer ** to the precipitate, and incubate at 37 for 2 hours. ** Lysis Buffer: 10 mm Tris-HCl, ph7.6, 10 mm EDTA, 50 mm NaCl, 0.5% SDS, 0.1 mg/ml Proteinase K 5. Add the same volume of Phenol/Chloroform/Isoamylalcohol, and vortex well. Centrifuge at 12,000 rpm for 5 min. 6. Collect the aqueous layer (upper) into a new tube. Add the same volume of Chloroform solution and vortex well. Centrifuge at 2,000 12,000 rpm for 5 min. 7. Collect the aqueous layer (upper) into a new tube. Add 50 μ l of 5 M NaCl and 0.9 ml of isopropanol. 8. Centrifuge at 12,000 rpm for 5 min, and collect the precipitate. 9. Rinse the precipitate with 70% ethanol, and then dry slightly. 10. Dissolve the precipitate with TE buffer and use it as DNA sample in the following PCR reaction. B. PCR 1. Mix the following reagents, and prepare them in 0.2 ml micro PCR tubes. per reaction 10 X Ex Taq Buffer (Mg 2+ plus) *1...5 μ l dntp Mixture (2.5 mm each) *1...4 μ l FLT3/ITD-1 Primer (10 pmol/ μ l)...1 μ l FLT3/ITD-2 Primer (10 pmol/ μ l)...1 μ l TaKaRa Ex Taq TM (5 units/ μ l) μ l Genome DNA * μ g Sterilized distilled water...up to 50 μ l Total 50 μ l *1: Supplied with TaKaRa Ex Taq TM *2: When Control Template is used, add in 1 μ l. 2. Perform PCR under the following condition sec sec. 35 cycles sec min. 3

4 Cat.# 6632 PCR FLT3/ITD Mutation Detection Set C. Agarose gel electrophoresis Perform electrophoresis by applying 5 μ l of PCR product with 3% NuSieve R 3:1 agarose gel in TAE buffer. Use a 100 bp DNA ladder as a molecular marker. After electrophoresis is completed, stain the gel with ethidium bromide and detect bands with a UV transilluminator. If the normal and mutant control templates are not clearly distinguishable from one another, it may be in improper electrophoresis conditions. It is recommended to determine the optimal conditions by adjusting the concentration of the gel or extending the electrophoresis time. Table.1 Amplified fragment size Sample Amplified size Normal samples 329 bp ITD mutant samples 329 bp(normal) & bp (ITD Mutant)* 3 Control Template (Normal) 329 bp Control Template (Mutant) 359 bp *3: Generally, two bands (normal at 329 bp and ITD around 347~419 bp) are detected with ITD mutant samples, but there may be cases in which only the ITD band is detected. D. Analysis with Agilent 2100 bioanalyzer Perform the analysis with 2100 bioanalyzer by following the instruction manual supplied with the instrument. 1. Dilute PCR products in TE buffer and prepare at ng/ μ l (almost 2-10 folds dilution). 2. Following the instruction manual of DNA 1000 LabChip Kit, prepare Gel-Dye Mix. 3. Set up the chip preparation stand and adjust the clip stopper. 4. Load Gel-Dye Mix on LabChip. 5. Add DNA marker and ladder into LabChip. 6. Add 1 μ l of the diluted PCR products into LabChip. 7. Mix for one minute with an exclusive mixer for LabChip. 8. Set LabChip into Bioanalyzer. Select DNA 1000 from the assay menu and start the analysis. The analysis of the maximum 12 samples completes around 30 minutes. 9. By referring the amplified fragment size in Table 1 above, compare the peak of sample with one of Control Template (Normal) on the bioanalyzer analysis chart. When the further peak is detected in longer chain is detected, the sample is as ITD mutation positive. VI. Example Experiment: A. Example of electrophoresis analysis [Methods] After PCR, electrophoresis with 3% NuSieve 3:1 agarose gel was performed to determine the size of the products. [Result] The band of normal sequence (329 bp) was verified with the Control Template (normal), and a 359 bp band was detected with the Control Template (mutant). For the DNA obtained from AML patients, a longer band approximately by 60 bp than the normal band (329 bp) was detected along with the normal band. This result indicates the presence of an ITD mutation. 4

5 PCR FLT3/ITD Mutation Detection Set Cat.# 6632 M bp 300 bp 359 bp 329 bp M: 100 bp DNA Ladder Marker 1: Genomic DNA from the bone marrow of an AML patient 2: Control Template (Mutant): 359 bp 3: Control Template (Normal): 329 bp Fig.1 Example of Control Template and AML patient's DNA by electrophoresis. B. Example using Agilent2100 Bioanalyzer [Method] PCR was by following the protocol specified in IV. Protocol. Analyzed samples are genomic DNA obtained from the bone marrow of an AML patient, Control Template (Control), and the Control Template (mutant). After PCR, the PCR products was applied to DNA 1000 LabChip Kit, and analyzed with Agilent 2100 bioanalyzer. [Results] The PCR products of the Control Templates (mutant) and (normal) were mixed and analyzed using the Agilent bioanalyzer. Two separate peaks were detected, indicating a normal peak corresponding to 329 bp and a mutant peak corresponding to 359 bp (Figure 2). For the genomic DNA obtained from the bone marrow of the AML patient, a peak corresponding to a DNA fragment longer approximately 60 bp than the 329 bp peak was detected along with the normal peak. This result indicates the presence of an ITD mutation (Figure 3). N M Horizontal: Migration time Vertical: Fluorescence N: Control Template (Normal) M: Control Template (Mutant) Figure 2. Example of Control Template using Agilent 2100 Bioanalyzer. 5

6 Cat.# 6632 PCR FLT3/ITD Mutation Detection Set N ITD Figure 3. Example of AML patient's genomic DNA using Agilent 2100 Bioanalyzer N: Normal ITD: ITD mutation XII. Reference: 1. Nakao M, Yokota S, Iwai T, et al (1996) Leukemia 10, Yokota S, Nakao M, Okuda T, et al (1997) Leukemia 11, Kiyoi H, Naoe T, Yokota S, et al (1997) Leukemia 11, Kiyoi H, Naoe T, Nakao Y, et al (2002) Blood 93, Kiyoi H, Naoe T (2002) Leukemia and Lymphoma 43, Zheng R, Friendman AD, Small D (2002) Blood 100, XIII. Note: This product is intended to be used for research purpose only. They are not to be used for drug or diagnostic purposes, nor are they intended for human use. They shall not to be used products as food, cosmetics, or utensils, etc. Takara products may not be resold or transfered, modified for resale or transfer, or used to manufacture commercial products without written approval from If you require licenses for other use, please call at or contact from our website at Assay results: assumes no responsibility for consequences resulting from the use of the analysis results performed for purposes other than research. [M31] FLT3/ITD mutation detection This product is covered by the claims of U.S. Patent No. 6,846,630 and its foreign counterpart patent claims. Agilent2100 bioanalyzer is a product of Agilent Technologies LabChip is a registered trademark of Caliper Technologies Corporation 6 Phone: Fax: