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Detection system of the A1075C mutation in the
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The purchase of this product does not provide a license to carry out patented applications. Kit Utility The kit analyzes the presence of a polymorphism in the position 1075 (A1075C) in the gene that encodes for the cytochrome P450 CYP2C9 (allele cyp2C9*3). Principle of the Assay The analysis for the detection of the A1075C mutation, requires a PCR amplification of the segment of the cyp2C9 gene (allele cyp2C9*3) that contains the mutation. The presence or absence of a base substitution is later detected by RFLP. There are three possible test results: Homozygote AA: when the A1075C mutation cannot be detected in neither of the gene alleles. Heterozygote AC: when the A1075C mutation is detected in only one of the gene alleles. Homozygote CC: when the A1075C mutation is detected in both alleles. Introduction Cytochrome P450 (CYP) 2C9 is a monooxigenase, which produces the hydroxylation of 16% the most commonly used drugs. It metabolizes different xenobiotics including S-warfarin, tolbutamide and phenytoin. The alteration of the CYP2C9 enzymatic activity might cause drug adjustment difficulties or toxicity problems. Several polymorphisms consisting in a single nucleotide substitution have been identified in the CYP2C9 gene. These polymorphisms determine metabolic phenotypes, which explain the individual and ethnical differences. Apart from the native protein CYP2C9*1, there exist at least five other allelic variants that produce alloenzymes with a reduced or deficient metabolic activity. Among Caucasian populations only the CYP2C9*2 and CYP2C9*3 variants are significant. Their allelic frequencies are 0.08-0.14 and 0.04-0.016, respectively. It has been described that those enzymes encoded by the CYP 2C9*2 (substitution C430T/ R144C) and CYP 2C9*3 (substitution A1075C/ I359L) allelic variants, have very low effectiveness in the in-vitro hydroxylation of warfarin.
Kit presentation: Color that identifies the kit: dark grey The kit includes: • 1 CYP2C9*3 Reaction Mix tube (light grey). • 1 CYP2C9*3 Restriction Enzyme tube. • 1 CYP2C9*3 DNA Positive Control, containing heterozygous control DNA (once
defrosted it is recommended to keep it at 4 ºC).
• 1 CYP2C9*3 Taq DNA polymerase tube. • 1 CYP2C9*3 Molecular Weight tube. It the amplification product band and all the
possible digestion bands. This tube should be kept in the post-amplification zone if possible.
Our kits are available in two sizes: 20 and 50 reactions. Necessary materials not provided with the test
• DNA-free PCR tubes • Suitable pipettes • Filtered pipette tips • Gloves and robe • Thermocycler • Cuvette for vertical electrophoresis • Acrylamide, electrophoresis buffer and loading buffer • Gel coloring system with silver nitrate. • Vortex • Bio-hazard disposable container
Precautions 1. Only for in-vitro use. 2. All samples, reagents and controls should be considered potentially infectious. 3. Do not use after the expiration date indicated on the package.
Storage and Stability The kit must be stored at -20ºC in order to assure its optimal performance through the expiration date indicated on the package. Specimen Characteristics The sample must be a DNA solution with a 50-100 ng / µl concentration, apt for PCR amplification. ATGen recommends obtaining the DNA from blood specimens by using ADN Facil kit. Protocol Pre-amplification zone: Defrost the reaction mix and shake it vigorously by vortexing. If possible, perform all manipulations in cold room. Preparation of the amplification mix:
• 1. Add 18 µl of reaction mix per sample to be tested. • 2. Add to the reaction mix, 1 µl of DNA Taq polymerase per sample to be
• 3. Homogenize by moderate vortexing or pipetting.
It is recommended to prepare a single amplification mix containing the necessary quantities of reaction mix and DNA Taq polymerase, according to the number of samples to be analyzed. It is necessary to add two reactions, one for the positive control and one for the negative control. Amplification:
• 4. Aliquot the amplification mix, dispensing 18 µl in properly labeled PCR
• 5. Add 2 µl of sample to each tube.
The samples must contain between 100 and 200 ng of DNA (We recommend to use ATGen´s ADN Facil Kit for the DNA extraction).
• 6. Add 2 µl of CYP2C9*3 DNA into the positive control tube and 2 µl of the
water previously used to dissolve the sample DNA in the negative control tube.
Program: 35 cycles at 94 ºC/0:30´, 58 ºC/0:30´, 72 ºC/0:30´; an initial denaturizing step: 5 minutes at 94 ºC and a final extension step: 5 minutes at 72ºC.
• 8. Place the tubes into the thermocycler when it reaches 94ºC.
Once the program has ended and in the case you are not going to immediately perform the next step, keep the tubes at 4ºC until the digestion stage. Optionally, the amplification can be tested by electrophoresis by loading 5 µl of the reaction product and using a 6% acrylamide gel. The expected size of the amplification product is 171 bp. Digestion:
• 9. Once the program has ended allow the temperature to drop until the tubes
reach room temperature and then add 1 µl of restriction enzyme to every amplification tube.
Obtention of test results 1. Prepare samples with the indicated quantity of an adequate loading buffer (e.g.
glycerol 30% p/v, xilencianol blue 0.25% p/v, bromophenol blue 0.25% p/v).
2. Load 5 µl of each digested amplification product and 5 µl of CYP2C9*3 molecular
3. Migrate the bromophenol blue (of the loading buffer): 8 cm in acrylamide.
4. Use silver nitrate for staining the acrylamide.
Note: After the digestion, the amplification product band must not be present. If this 171 bp band appears, it means that a partial digestion has occurred.
10 % acrylamide gel stained with silver nitrate showing the following results: 1: AA homozygote individual. 2: AC heterozygote individual. The control DNA must show this result. MW: CYP2C9*3 molecular weight marker with the amplification band and the possible digestion bands. Bibliography 1. Lancet 1999; 353: 717-719 2. J.A.M.A. 2002; 287: 1690-1698
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