Warfarin Genotyping (VKORC1) Kit (for Academic Instructions) Product # 53900

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1 3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: (905) Fax: (905) Warfarin Genotyping (VKORC1) Kit (for Academic Instructions) Product # Product Insert Warfarin is an anticoagulation drug that is used in the prevention of thrombosis. It functions to decrease blood coagulation by inhibiting vitamin K epoxide reductase, thus antagonizing vitamin K 1 recycling and subsequently depleting active vitamin K 1. It is commonly applied to prevent or treat various cardiac arrhythmia such as atrial fibrillation as well as pulmonary embolism. Dosing of Warfarin is delicate as its major adverse effect is hemorrhage or bleeding. This is further complicated by its interaction with many commonly used medications as well as various food products. Hence at the initial stage of a Warfarin treatment, the patient may be subjected to multiple blood testing such that a proper Warfarin dosage can be achieved. The effectiveness of Warfarin treatment is also affected partially by genetic factors. In particular, pharmacogenomic studies have identified three single nucleotide polymorphisms (SNPs) that are an important determinant of the initial Warfarin dosage. Two of the SNPs are found in the cytochrome P450 enzyme gene CYP2C9 known as CYP2C9*2 (C430T) and CYP2C9*3 (A1075C). The third SNP is found in the Vitamin K Epoxide Reductase Complex Subunit 1 (VKORC1) known as VKORC1 (1639G A). Several reports suggest that using such genetic information may improve the initial estimate of what is a reasonable warfarin dose for individual patients. For example, mutations of VKORC1 make it less susceptible to suppression by warfarin and hence individuals carrying such mutations may require higher dosage of warfarin. Principle of the Test and Product Description Norgen s Warfarin Genotyping (VKORC1) Kit uses PCR to determine the genotype of a human Vitamin K Epoxide Reductase Complex Subunit 1 (VKORC1) polymorphism (a G A mutation). First, genomic DNA is isolated from the individual(s). This can be achieved by using Norgen's Saliva DNA Collection, Preservation and Isolation Kit (Cat# RU35700). The genomic DNA is then subjected to two PCR reactions - one for the detection of normal (Control) version (G) of the locus and one for the detection of the altered (Mutation) version (A). The products from the two PCR reactions are then resolved and visualized using standard DNA agarose gel electrophoresis. The expected molecular weights of both PCR products are identical, at 377 bp. The presence or absence of the PCR product is then recorded. The information will then be used to determine the genotype of each individual for VKORC1, with the possibility of being Homozygous Wild Type (PCR product only present in Wild Type reaction), Heterozygous (PCR product present in both reactions) or Homozygous Mutant (PCR product only present in Mutant reaction). Kit Components: Component Contents 2x PCR Master Mix with Dual Dye 0.5 ml 10x VKORC1 Control PCR Primer Mix (G) 30 μl 10x VKORC1 Mutation PCR Primer Mix (A) 30 μl Nuclease-Free Water 1.25 ml Positive Control DNA 10 μl PCR Sizer 100 bp DNA Ladder 100 μl 10x TAE 100 ml Agarose 5 g Product Insert 1 a The positive control is purified DNA fragments.

2 Recommended Product for DNA Isolation (not included in the kit): Saliva DNA Collection, Preservation and Isolation Kit (Norgen Biotek Cat# RU35700) Customer-Supplied Reagents and Equipment Disposable powder-free gloves Benchtop microcentrifuge Micropipettors Sterile pipette tips with filters PCR Strips and Caps Thermocycler / PCR Machine Agarose Gel Electrophoresis Apparatus and Power Supply (Optional) 10 mg/ml Ethidium Bromide (Cat# 28033) Storage Conditions and Product Stability The 2x PCR Master Mix with Dual Dye, 10x VKORC1 Control PCR Primer Mix, 10x VKORC1 Mutation PCR Primer Mix, and Positive Control DNA should be kept tightly sealed and stored at -20 o C. These can be stored for up to 1 year without showing any reduction in performance. Repeated thawing and freezing (> 2 x) of these reagents should be avoided, as this may reduce the sensitivity. If the reagents are to be used only intermittently, they should be frozen in aliquots. All other kit components should be kept tightly sealed and stored at room temperature (15-25 o C). All kit components can be stored for up to 1 year without showing any reduction in performance. General Precautions The user should exercise the following precautions when using the kit: Use sterile pipette tips with filters. Store and extract positive material (specimens, controls and amplicons) separately from all other reagents and add it to the reaction mix in a spatially separated facility. Thaw all components thoroughly at room temperature before starting an assay. When thawed, mix the components and centrifuge briefly. Work quickly on ice. Quality Control In accordance with Norgen s ISO 9001 and ISO certified Quality Management System, each lot of Norgen s Warfarin Genotyping (VKORC1) Kit, including 2x PCR Master Mix with Dual Dye, 10x VKORC1 Control PCR Primer Mix, 10x VKORC1 Mutation PCR Primer Mix, and Positive Control DNA are tested against predetermined specifications to ensure consistent product quality. Product Warranty and Satisfaction Guarantee NORGEN BIOTEK CORPORATION guarantees the performance of all products in the manner described in our product manual. The customer must determine the suitability of the product for its particular use. Safety Information Wear gloves when performing the protocol. Refer to the MSDS sheets for safety procedures regarding reagents used.

3 Protocol Summary of Method This protocol is a multi-part procedure involving: 1. Collection and Purification of Saliva DNA from the individual. 2. PCR Amplification of the normal and mutated version of the VKORC1 locus from purified Saliva DNA. 3. Resolution of PCR fragments on an agarose gel. 4. Genotyping of individual based on PCR results. Section 1. Collection and Purification of Genomic DNA (Saliva) from Individual of Interest Notes: The recommended input source for DNA is Saliva. Additional guidelines for sample preparation are provided below. Other non-invasive inputs (such as buccal swab) could be used. However, the quality and the length of sequence read could vary, depending on the purity and the integrity of the genomic DNA isolated from these samples. Recommended Products: Norgen s Saliva DNA Collection, Preservation and Isolation Kit (Cat# RU35700) - Contains Saliva Collection and Preservation Devices, as well as reagents for alcoholprecipitation-based DNA purification. Norgen s Saliva DNA Isolation Kit (Cat# RU45400) - Provides reagents for column-based DNA purification from saliva samples collected using Norgen s Saliva DNA Collection, Preservation and Isolation Kit (Cat# RU35700). Recommended Procedure: 1. Perform saliva collection and DNA isolation using Norgen s Saliva DNA Collection, Preservation and Isolation Kit (Cat# RU35700). Follow the procedure for extraction of DNA from 0.5 ml of preserved saliva. 2. The average expected yield of DNA from 0.5 ml preserved saliva is ~20 μg. The average expected OD 260/280 ratio is ~1.70. This could be verified using a standard spectrophotometer. 3. Based on the DNA concentration of the sample, dilute the DNA to approximately 10 ng/μl using nuclease-free water or TE. Proceed to Section 2. Section 2. PCR Amplification of the Normal and Mutated VKORC1 Notes: Before use, suitable amounts of all PCR components should be completely thawed at room temperature, vortexed and centrifuged briefly. The amount of 2x PCR Master Mix with Dual Dye, 10x VKORC1 Control PCR Primer Mix, 10x VKORC1 Mutation PCR Primer Mix and Positive Control DNA provided is enough for tests of up to 8 individuals (8 reactions for control VKORC1 detection and 8 reactions for mutant VKORC1 detection, plus Positive and Negative Controls). For each DNA sample, one PCR reaction using the 10x VKORC1 Control PCR Primer Mix and one PCR reaction using 10x VKORC1 Mutation PCR Primer Mix should be set up. A Positive Control DNA is included and could be applied directly to the procedure (see below). It is highly recommended that sterile pipette tips with filters be used to avoid any crosscontaminations.

4 1. Thaw the 2X PCR Master Mix with Dual Dye and the PCR Primers completely. Leave the thawed mixes on ice if the reaction is not set up immediate. 2. Set up 20 μl Control PCR Reactions, using the 10x VKORC1 Control PCR Primer Mix, as described in Table 1 below. Table 1. Sample PCR Reaction Set up for VKORC1 Control PCR Components Nuclease-free Water 2X PCR Master Mix 10x VKORC1 Control PCR Primer Mix (G) Volume Reserved for DNA (Concentration at ~ 10 ng/ μl) Total Per 20 μl reaction 7 μl 10 μl 2 μl 1 μl 20 ul 3. Add each component (except the DNA template) of the PCR reaction, in the order shown in Table 1 into the PCR tube. Leave the tube, capped, on ice. 4. Add 1 μl of DNA to the PCR reaction mix. 5. Cap the PCR tube. Mix briefly. Label the PCR tube such that each test individual could be easily identified. 6. Repeat the set up for all test individuals. Leave the tubes, capped, on ice. 7. Set up 20 μl Mutation PCR Reactions, using the 10x VKORC1 Mutation PCR Primer Mix, as described in Table 2 below. Table 2. Sample PCR Reaction Set up for VKORC1 Mutation PCR Components Nuclease-free Water 2X PCR Master Mix 10x VKORC1 Mutation PCR Primer Mix (A) Volume Reserved for DNA (Concentration at ~ 10 ng/ μl) Total Per 20 μl reaction 7 μl 10 μl 2 μl 1 μl 20 ul 8. Add each component (except the DNA template) of the PCR reaction, in the order shown in Table 2 into the PCR tube. Leave the tube, capped, on ice. 9. Add 1 μl of DNA to the PCR reaction mix. 10. Cap the PCR tube. Mix briefly. Label the PCR tube such that each test individual could be easily identified. 11. Repeat the set up for all test individuals. Leave the tubes, capped, on ice. 12. For each PCR run, Positive Controls (PosC) and Negative Controls (NegC) should be set up.

5 13. Set up Positive Controls, one using the 10x VKORC1 Control PCR Primer Mix and one using 10x VKORC1 Mutation PCR Primer Mix as described in Table 3. Table 3. Positive Control PCR Reaction Set up (One for VKORC1 Control, One for VKORC1 Mutation) Components Nuclease-free Water 2X PCR Master Mix 10x VKORC1 Control PCR Primer Mix (G) or 10x VKORC1 Mutation PCR Primer Mix (A) Positive Control DNA Total Per 20 μl reaction 7 μl 10 μl 2 μl 1 μl 20 ul 14. Add each component (except the PosC) of the PCR reaction, in the order shown in Table 3 into the PCR tube. Ensure one tube set for Control PCR and one tube set for Mutation PCR. Leave the tube, capped, on ice. 15. Add 1 μl of PosC to each of the PCR reaction mix. 16. Cap the PCR tube. Mix briefly. Label the PCR tube such that the Positive Controls could be easily identified. Leave the tubes, capped, on ice. 17. Set up Negative Controls, one using the 10x VKORC1 Control PCR Primer Mix and one using 10x VKORC1 Mutation PCR Primer Mix as described in Table 4. Table 4. Negative Control PCR Reaction Set up (One for VKORC1 Control, One for VKORC1 Mutation) Components Nuclease-free Water 2X PCR Master Mix 10x VKORC1 Control PCR Primer Mix (G) or 10x VKORC1 Mutation PCR Primer Mix (A) Total Per 20 μl reaction 8 μl 10 μl 2 μl 20 ul 18. Add each component of the PCR reaction, in the order shown in Table 4 into the PCR tube. Ensure one tube set for Control PCR and one tube set for Mutation PCR. 19. Cap the PCR tube. Mix briefly. Label the PCR tube such that the Negative Controls could be easily identified. 20. Load all samples into a thermocycler. Set up the PCR Cycling Time as below: 94 C for 2 minutes 1 Cycle 94 C for 10 seconds 60 C for 30 seconds 40 Cycles 72 C for 45 seconds 72 C for 10 minutes 1 Cycle Hold for 4 C

6 21. Proceed to Section 3 to resolve the PCR products on an agarose gel for visualization. Otherwise, store the reactions at -20 o C. Section 3. Resolution of the Normal and Mutated VKORC1 PCR Products by Agarose Gel Electrophoresis Notes: Resolved DNA gels could be visualized by staining with Ethidium Bromide (EtBr). Caution: EtBr is a known mutagen. DNA gels stained with EtBr are to be visualized under ultraviolet (UV) light. Wear a lab coat, eye protection and gloves when working with this chemical. Alternatively, the DNA gel can be stained with a non-mutagenic stain and visualized according to its excitation/emission spectrum. A. Pouring a Standard 2 % (W/V) Agarose Gel 1. Prepare 1 L of 1x TAE by diluting 100 ml of 10x TAE with 900 ml of deionized water. 2. Pour 100 ml of the 1x TAE into a microwavable flask. The remaining 900 ml 1x TAE could be set aside and used as running buffer for electrophoresis. 3. Measure 2 g of agarose. 4. Pour the agarose powder into the microwavable flask containing the 100mL of 1xTAE. 5. Microwave for 1 to 3 minutes until the agarose is completely dissolved. Use a hot plate for boiling if a microwave is not available. Note: Caution HOT! Be careful when stirring, as eruptive boiling can occur. The most appropriate way to melt the agarose is to microwave for seconds, stop and swirl, and then continue towards a boil. Watch the flask closely as it has a tendency to boil over. 6. Allow the melted agarose solution to cool down for around 10 minutes. 7. (Optional) For Ethidium Bromide (EtBr) visualization, add 1 μl of 10 mg/ml Ethidium Bromide (EtBr) to the melted agarose solution and mix by swirling gently. Note: Caution: EtBr is a known mutagen. Wear a lab coat, eye protection and gloves when working with this chemical. Note: An alternative non-mutagenic stain could be used. Mix the stain with the agarose solution according to manufacturer's recommendation. 8. Pour the agarose into a gel tray with the well comb in place. Note: Ensure that there are enough wells for the PCR products as well as an additional 5 lanes for Positive Control (2 wells), Negative Control (2 wells) and molecular weight ladder (1 well) 9. Let the newly poured gel sit at room temperature for minutes, until it has completely solidified.

7 B. Loading Samples and Running the DNA Agarose Gel 1. Once solidified, place the agarose gel into the appropriate gel box. Orient the gel in such a way that the loading well is closest to the negative electrode. Note: DNA is negatively charged and will run towards the positive electrode. In most gel boxes, the negative electrode is black while the positive electrode is red. 2. Fill the gel box with the remaining 1xTAE prepared in Step A2 above. Pour enough buffer such that it covers about 5 mm above the top of the gel. Note: Depending on the size of the gel box, additional amount of 1x TAE may have to be prepared. 3. Carefully load 10 μl of the DNA molecular weight ladder (PCR Sizer, provided) into the first lane of the gel. Note: The DNA ladder is already pre-mixed with a blue loading dye. 4. Carefully load 15 μl of the Control PCR product of the first test individual into the well immediately next to the DNA ladder. Note: The PCR reaction is already pre-mixed with an orange loading dye. 5. Carefully load 15 μl of the Mutation PCR product of the first test individual into the well immediately next to the Control PCR product. Note: The PCR reaction is already pre-mixed with an orange loading dye. 6. Continue to load the remaining wells of the gel with the Control and Mutation PCR products (15 μl each) of the rest of the test individuals. Ensure that the two PCR products of the same individual are loaded side-by-side. 7. Load 15 μl of the Control and Mutation PCR product of the Positive Control. 8. Load 15 μl of the Control and Mutation PCR product of the Negative Control. 9. Connect the gel box with the power supply appropriately. Connect the negative charge outlet of the power supply to the negative (black) electrode. Connect the positive charge outlet to the positive (red) electrode. 10. Set the power supply Voltage at constant at 150 V. Note: The current (Amp) will be variable. It could be set at the maximum amount available. 11. Start to run the gel at 150V for about 45 minutes Note: The loading dye of the PCR mix will be separated into two bands - the bigger band will have a dark red color (Cresol-Red, ~ 1 kbp) while the smaller band will have a light orange/yellow color (Orange G, ~ bp). The actual PCR products of VKORC1 migrate between these two bands. Hence, the two colored band could be used to track the movement of the PCR product. For the most optimal condition, allow the Orange G dye (light orange/yellow band) to migrate ~ 75-80% of the way down the gel. 12. Turn OFF power, disconnect the electrodes from the power supply, and then carefully remove the gel from the gel box. Note: It is desirable to place the gel in a suitable transport container or tray. 13. Perform the appropriate staining for DNA visualization. For EtBr Staining, use any device that has UV light to visualize your DNA fragments. Capture a picture of the DNA gel for record. Note: When using UV light, protect your eyes and skin by wearing safety goggles or a face shield, gloves and a lab coat.

8 C. Analyzing the Gel 1. Print out a copy of the picture of the DNA gel. 2. Label each lane appropriately. Clear indicate the DNA molecular weight ladder, the Control and Mutation PCR products of each test individual, Positive Control and Negative Control. The PCR products have a molecular weight of 377 bp. 3. An example of a test gel is shown below: AA AA GG GA PosC NegC MW Homozygous Wild Type = GG Homozygous Mutant = AA Heterozygous = GA Figure 1. Rapid Genotyping of Human VKORC1 Single Nucleotide Polymorphism by End-Point PCR. Fifteen micro-liter of PCR reactions, one specific for detecting control/wild-type (WT) VKORC1 (denoted by the base "G") and one specific for detecting mutated (SNP) VKORC1 (denoted by the base "A"), were loaded on a 1X TAE 2 % Agarose DNA gel with 10 μl of Norgen's PCR Sizer 100 bp DNA Ladder (Cat# 11400) as a MW Marker (150V for 45 minutes). The expected molecular weight of the PCR product is 377 bp. The presence or absence of the PCR product in each reaction helps to determine the genotype of each individual for VKORC1, as indicated in the figure. 4. Record the genotyping results of the test in the table provided below.

9 VKORC1 Genotyping Results Individual ID VKORC1 Control Detection VKORC1 Mutation Detection Genotype of Individual Does this individual have a VKORC1 mutation? Predicted Warfarin dosage requirement (Enter "G" for detection and leave blank for no detection) (Enter "A" for detection and leave blank for no detection) (Combine the two detection columns. Possible outcomes are "GG", "GA" and "AA") (Y/N) (Normal or Higher) PosC NegC

10 Related Products Product # Saliva DNA Collection, Preservation and Isolation Kit RU mg / ml Ethidium Bromide Technical Assistance NORGEN s Technical Service Department is staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of NORGEN products. If you have any questions or experience any difficulties regarding Norgen s Warfarin Genotyping (VKORC1) Kit or NORGEN products in general, please do not hesitate to contact us. NORGEN customers are a valuable source of information regarding advanced or specialized uses of our products. This information is helpful to other scientists as well as to the researchers at NORGEN. We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques. For technical assistance and more information, please contact our Technical Support Team between the hours of 8:30 and 5:30 (Eastern Standard Time) at (905) or Toll Free at or call one of the NORGEN local distributors ( or through at techsupport@norgenbiotek.com Schmon Parkway, Thorold, ON Canada L2V 4Y6 Phone: (905) Fax: (905) Toll Free in North America: Norgen Biotek Corp. PI