Novallele Genotyping Assay User Guide

Transcription

1 October 2017 Assay User Guide Copy Number A complete set of instructions to determine copy number variation using the Novallele copy number reagents For Research Use Only. Not for use in diagnostic procedures.

2 Canon BioMedical is focused on empowering the biomedical research and healthcare communities through innovative technologies and solutions. Our solutions will enable clinicians and scientists to improve our health and advance science. Canon BioMedical pursues innovation in harmony with Canon's Kyosei philosophy ---- defined as "all people, regardless of race, religion, or culture, harmoniously living and working together into the future." Page 2 of 24 Novallele Genotyping Assay User Guide

3 Contents Introduction 5 Summary and Explanation 5 Principle of the Procedure 6 Sample type and DNA extraction 7 Controls 8 SMN1 controls 8 SMN2 controls 9 Thermocycler 9 PCR run 10 Materials Available from Canon BioMedical 11 Materials Required but Not Available from Canon BioMedical 11 Warnings and Precautions 12 Reagent Storage and Handling 12 Novallele copy number assays 12 Novallele Copy Number Mastermix 12 Procedure 13 Important points before starting 13 Things to do before starting 13 Novallele Genotyping Assay User Guide Page 3 of 24

4 DNA extraction 13 DNA quality 14 PCR setup 14 PCR run 16 Data Analysis 16 Troubleshooting 18 Contact Information 19 Ordering Information 20 Page 4 of 24 Novallele Genotyping Assay User Guide

5 Introduction Rapid PCR with high-resolution melting (HRM) analysis enables scientists to quickly determine copy number variations for their genes of interest. We developed a functionally-verified chemistry that supports faster ramping rates and shortened cycling times as compared to typical PCR conditions. With a single reaction, you can determine copy number---- no sample replicates required. This guide provides instructions to determine copy number variation using the Novallele copy number reagents and an HRM-enabled thermocycler. For Research Use Only. Not for use in diagnostic procedures. Summary and Explanation Changes to germline DNA can occur in many different forms, including single-nucleotide polymorphisms (SNPs), epigenetic modifications, chromosomal rearrangements, and aneuploidies. Additionally, copy number variation, which can affect either a section of a gene or an entire gene, exists throughout the genome. Deletions and duplications of certain genes are linked to drug metabolism, cancer, and numerous inherited disorders. To analyze copy number variations, numerous technologies exist such as the comparative C T (ΔΔC t ) method, digital PCR, fluorescent in-situ hybridization, microarrays, multiplex ligation-dependent probe amplification, and next-generation sequencing. The variety of methods enables researchers to match the correct technology with not only their experimental question but also user preferences for cost, data analysis, and turnaround time. Novallele Genotyping Assay User Guide Page 5 of 24

6 Principle of the Procedure The Novallele copy number assays, in combination with the Novallele Copy Number Mastermix, contain all the reagents needed to determine the copy number of a human genomic DNA (gdna) sample. The Novallele copy number assays and Novallele Copy Number Mastermix are used with a thermocycler to perform rapid PCR followed by high-resolution melting (HRM) analysis. To determine copy number variations for the gene of interest, the Novallele copy number assays compare the relative fluorescence values of samples to the fluorescence of characterized controls. Analyzing the relative fluorescence of samples is a simple, fast, and sensitive method to determine copy number (1). To generate the relative fluorescence data required, the gene of interest and an internal reference, which is conserved at two copies, are both amplified and their fluorescence captured using HRM. With deoxynucleotide triphosphates (dntps) as the limiting reagent in the PCR, the gene of interest and internal reference compete against one another in the reaction mixture. Samples with duplications in the gene of interest will preferentially amplify over the internal reference since the gene of interest has more primer binding sites; inversely, the internal reference will preferentially amplify over samples with deletions in the gene of interest since the internal reference has more primer binding sites. In order to keep the fluorescence values relative between the amplified products, the PCR is stopped during the linear amplification stage. This is accomplished by reducing the number of PCR cycles to 25. After the PCR and HRM analysis are complete, the data are normalized for fluorescence output and corrected for any shifts in melting temperature. Once the normalization steps are complete, copy number variation is determined. The clustering of samples with characterized controls in the difference plot determines the copy number of the sample. 1 Zhou, L., Palais, R. A., Paxton, C. N., Geiersbach, K. B. and Wittwer, C. T. (2015) Copy number assessment by competitive PCR with limiting deoxynucleotide triphosphates and high-resolution melting. Clin Chem 61, Page 6 of 24 Novallele Genotyping Assay User Guide

7 An overview of the copy number protocol: Sample type and DNA extraction We tested the Novallele copy number assays with gdna extracted from cell lines and blood. Sample preparation is critical to accurate downstream analysis. Make sure to select a DNA extraction method suitable for your application. Use any method that yields acceptable concentration, purity, and integrity of DNA, but make sure to validate the DNA extraction method (see DNA Quality). Maintain the selected DNA extraction method throughout the experiment as changes in salt concentration and elution buffer components can affect sample analysis. It is essential that all samples are prepared using the same method to reduce variability in the subsequent analysis. Recommendation: Freeze extracted DNA samples to store if not performing the experiment at a later date. DO NOT use extracted DNA samples that are older than one month as data variability may occur when extracted DNA is stored for extended periods. We have achieved comparable results with the following kits: Roche MagNA Pure Compact Nucleic Acid Isolation Kit I QIAGEN QIAamp DNA Blood Mini Kit Promega Maxwell RSC Blood DNA Kit Promega Maxwell 16 Blood DNA Purification Kit Novallele Genotyping Assay User Guide Page 7 of 24

8 Controls We recommend routinely using negative controls in your experiments, and each run must include a set of characterized controls for different copy numbers. In order to determine a sample s copy number, the sample s melt curves are compared to the melt curves of the characterized controls included in the run. For each run, include at least one no-template control (NTC) to rule out environmental contamination. To prepare the NTC reaction, substitute nuclease-free water instead of the sample. We obtained characterized controls from Coriell Institute for our experiments. We are providing the following information to assist you with acquiring characterized controls. You can also use other controls with the Novallele copy number assays if you are confident in the characterization of these controls. A 1-copy SMN2 control is not included in the following list. We created the control by combining the 0-copy control with the 2-copy control at a 1:1 ratio. Website: Phone: SMN1 controls SMN1 copy number Coriell Institute catalog number 0 NA NA NA NA17117 Page 8 of 24 Novallele Genotyping Assay User Guide

9 SMN2 controls SMN2 copy number Coriell Institute catalog number 0 NA NA NA NA03814 Thermocycler Using a thermocycler with HRM capabilities, the sample DNA is amplified by PCR and followed by amplicon melting. During the PCR, a fluorescent dye at saturating concentrations binds to doublestranded DNA. The critical element of HRM is that the dye is specific and sensitive -- it only fluoresces when bound to double-stranded DNA. The optical system of the HRM-enabled thermocycler captures the reduction in fluorescence intensity as double-stranded DNA denatures into single strands. We developed and verified the performance of the Novallele copy number assays and Novallele Copy Number Mastermix on the Applied Biosystems QuantStudio 6, Bio-Rad CFX Connect, PCRmax Eco 48, QIAGEN Rotor-Gene Q, and Roche LightCycler 480 instruments. Other HRM-enabled thermocyclers such as the BioFire LS32 and Roche LightCycler 2.0 may be compatible; however, we have not tested the Novallele copy number assays and Novallele Copy Number Mastermix on these instruments. When using a Applied Biosystems QuantStudio, make sure run a calibration plate using LC green dye. Use the recommended calibration kit with the protocol specified by the manufacturer before running any samples using the Novallele copy number assays. If necessary, please contact Canon BioMedical at 844-CANONBIO for additional support. Novallele Genotyping Assay User Guide Page 9 of 24

10 PCR run The Novallele copy number assays in combination with the Novallele Copy Number Mastermix contain all the reagents needed to determine copy number of a human gdna sample. Samples containing DNA are mixed with the Novallele copy number assay and Novallele Copy Number Mastermix in either a microcentrifuge tube or a well plate. Each Novallele copy number assay contains enough reagent for 200 reactions if using the recommended amount of 4.0 µl per 10 µl reaction. For each Novallele copy number assay, a product specification sheet is provided that contains the assay conditions for programming your HRM-enabled thermocycler. The product specification sheets are included with each Novallele copy number assay and can also be found at Important: The Novallele Copy Number Mastermix is essential for the success of the Novallele copy number assays. DO NOT replace the Novallele Copy Number Mastermix with other products. Page 10 of 24 Novallele Genotyping Assay User Guide

11 Materials Available from Canon BioMedical This user guide applies to multiple products that are ordered individually from Canon BioMedical as described in the table below. Product name Catalog number Number of reactions Amount (µl) Novallele Copy Number Mastermix SMN1 Novallele Copy Number Assay SMN2 Novallele Copy Number Assay Materials Required but Not Available from Canon BioMedical Important: Make sure all instruments are calibrated according to the manufacturer s recommendations. HRM-enabled PCR thermocycler 96-well or 384-well plate or applicable thermocycler reaction vessels, such as PCR tubes or capillaries Spectrophotometer and associated consumables such as cuvettes, etc. Nuclease-free water Single-channel or multichannel pipettes with nuclease-free tips Nuclease-free microcentrifuge tubes Centrifuge and rotor for well plate Novallele Genotyping Assay User Guide Page 11 of 24

12 Warnings and Precautions When working in a laboratory, always wear a lab coat, disposable gloves, and protective eyewear. Always follow your national and local safety policies and procedures regarding the appropriate handling and disposal of biological and chemical materials. For more information, please consult the applicable safety data sheets (SDSs). To obtain an SDS for our products, visit or contact us at 844-CANONBIO ( ). Reagent Storage and Handling When stored correctly, the materials described below are stable for 6 months from the date of receipt. If your products arrive damaged, please contact us at 844-CANONBIO ( ). Novallele copy number assays Store the Novallele copy number assays at C. Novallele Copy Number Mastermix IMPORTANT: Store the Novallele Copy Number Mastermix away from light. Store the Novallele Copy Number Mastermix at C and away from light as the reagent is lightsensitive. Page 12 of 24 Novallele Genotyping Assay User Guide

13 Procedure Important points before starting Use sterile aerosol barrier pipette tips designed for PCR applications. Do not use Diethylpyrocarbonate (DEPC)-treated water. Use high-quality, nuclease-free water. Novallele reagents can be stored during test preparation for up to two hours at room temperature ( C). Things to do before starting Familiarize yourself with your HRM-enabled thermocycler before starting this protocol. Refer to the applicable user manual and follow the manufacturer s instructions for proper operation and maintenance. Pipetting accuracy and precision affect the consistency of results. Make sure that all pipettes and instruments have been checked and calibrated according to the manufacturer s recommendations. Set up two physically separate workspaces; one for PCR setup and another for PCR and post- PCR processing operations. The physical separation will minimize amplicon contamination that may impact results and data interpretation. Decontaminate PCR workspaces and labware (pipettes, tube racks, etc.) with UV light and % bleach followed by wiping with 70% isopropanol. Thaw frozen gdna samples thoroughly at room temperature ( C) or on ice before starting. DNA extraction Extract the DNA from your sample. For guidance on DNA extraction, see Sample type and DNA extraction, page 7. Novallele Genotyping Assay User Guide Page 13 of 24

14 Important: Prepare all samples using the same extraction method to reduce variability in the subsequent analysis. HRM is sensitive to changes in salt and buffer components, and using the same extraction method reduces variability in the subsequent analysis. DNA quality After DNA extraction is complete, we recommend determining the purity and concentration of the DNA samples. DNA samples must contain fragments of at least 150bp for PCR-based analysis. Determine the purity of all DNA samples using UV spectrophotometric measurements or related methods. The acceptable A 260 :A 280 range for samples is The concentration, as measured by A 260, should be greater than or equal to 10 ng/µl. Exclude samples with a concentration of less than 10 ng/µl. PCR setup 1. Mix the reagents by briefly inverting or vortexing the Novallele copy number assay and Novallele Copy Number Mastermix tubes. 2. Centrifuge the Novallele copy number assay and Novallele Copy Number Mastermix tubes for seconds. 3. Prepare a reaction for each sample and control according to the table below by combining the specified components in a microcentrifuge tube for each sample or control. Important: For each thermocycler run, include a characterized control for each copy number that can be differentiated. Characterized controls use the same amount of DNA and are prepared the same as a sample. One characterized control reaction per run is sufficient for determining copy number of the samples included in the run. See Controls, page 8, for additional information. Recommendation: For each thermocycler run, include at least one NTC prepared by substituting nuclease-free water for the sample. Page 14 of 24 Novallele Genotyping Assay User Guide

15 Notes: The calculations below include 20% more than the required amount to prevent having an insufficient reaction volume for the run. The volume of sample is based on a DNA concentration of 10 ng/µl. If using a sample with a different DNA concentration, make sure to add 40 ng of DNA per reaction and adjust the amount of nuclease-free water. Table 1. Reaction preparation. Reagent One reaction Novallele Copy Number Mastermix 4.0 µl Novallele copy number assay 4.0 µl Sample or characterized control at 10 ng/µl 4.0 µl Total 12.0 µl 4. Vortex each tube for seconds. Centrifuge briefly. 5. Add 10 µl of the reaction to each well of the applicable vessel for the thermocycler (96-well plate, 384-well plate, capillary, etc.) using a single-channel pipette. Important: Make sure to change pipette tips between each reaction to avoid cross-contamination between the wells. 6. Tightly seal the applicable vessel as directed by the manufacturer. 7. Make sure that bubbles are not present in the wells of the plate. If bubbles are present, centrifuge the well plate at 1000 rpm for one minute for a 96-well plate or 2000 rpm for two minutes for a 384-well plate. Note: This is not required for QIAGEN Rotor-Gene Q or BioFire LS32 instruments. Novallele Genotyping Assay User Guide Page 15 of 24

16 8. Program the HRM-enabled thermocycler according to the conditions on the product specification sheet provided with the Novallele copy number assay. Note: The product specification sheet for each assay is available at 9. Place the applicable vessel in the thermocycler. If recommended by the manufacturer, use a compression pad with the optical film-sealed plate/disc formats. PCR run 1. Start the PCR run. Important: Do not open any processed plate. Removing the strips or film releases the amplicon into the air and may cause contamination of future PCR runs. 2. After the PCR run is finished, visually inspect each well for any sign of evaporation. If evaporation is observed, note the evaporated wells as this may affect the results of data analysis. 3. Dispose of the applicable vessel. Data Analysis Using the difference plot of the melt curves, the copy number of a sample is determined by comparing the fluorescence patterns of the sample to the characterized controls. Before determining copy number, smooth melting curves are used to normalize sample fluorescence as means to correct for plate positioning, different DNA input levels, or other amplification variables that affect fluorescence output. Subtle shifts in melting temperature can arise from different buffer compositions in each sample. To cluster samples and controls, the melting temperatures of all samples and controls must align. The derivative plot is used to correct the melting temperature of the PCR products. Once melting temperatures are corrected, the difference plot is used to determine copy number. A baseline cluster that is normally a 2-copy characterized control is selected. The characterized Page 16 of 24 Novallele Genotyping Assay User Guide

17 controls and samples clustering with the baseline copy number are set to a near-zero value. Characterized controls and samples with fewer than two copies are in discrete clusters below the baseline; characterized controls and samples with more than two copies are clustered above the baseline. The following difference plot distinguishes copy numbers using a 1-copy characterized control as the baseline (blue lines). Analyze your data using the Novallele HRM Analyzer. The Novallele HRM Analyzer is free, web-based software for HRM analysis. The software is available at the following website: Refer to Novallele HRM Analyzer User Guide for instructions on determining the copy number of your samples. If you require assistance analyzing your results, please visit our website at us at contactus@canon-biomedical.com, or call us at 844-CANONBIO ( ). Novallele Genotyping Assay User Guide Page 17 of 24

18 Troubleshooting Hopefully you do not have any problems with your Novallele products, but this section provides helpful hints from the scientists that developed the Novallele products. Additional information and content can also be found at If you require assistance beyond these resources, please us at or call us at 844-CANONBIO ( ). How do I analyze my data? Use the Novallele HRM Analyzer to analyze your results. The free, web-based software is found at the following website: The PCR protocol defines the amplification cycle number as 25 cycles. Why is the cycle number less than normal for a PCR protocol? The amplification cycle number is less than normal so that the PCR can be analyzed at a point that the PCR product amount is proportional to the copy number. The derivative plot for my sample has one or two melting peaks. What are the peaks? All Novallele copy number assays are designed as a duplex PCR. Each PCR assay contains a primer pair for the target as well as a primer pair for a conserved 2-copy reference gene. The melt peak at the lower temperature area of the graph is the PCR product of the gene of interest; however, the lower peak may not always present. When the sample contains zero copies of the gene, there is not a peak in the lower temperature region. The other peak in the higher temperature region of the graph is the PCR product from the reference peak. Page 18 of 24 Novallele Genotyping Assay User Guide

19 The no-template control (NTC) was amplified in my PCR. What should I do? NTC amplification is usually caused by reagent or DNA contamination. We recommend running the assay again with new reagents. Follow good laboratory practices for preventing PCR contamination. The sample difference plots do not match with the characterized controls. What should I do? Do not be alarmed if your sample copy number curve is different when compared to the characterized controls. A small difference between the melt curves is likely due to the software, DNA quality, well location, or sample preparation. The amplitude of the sample melt cures can be slightly above or below the characterized control copy number but should remain within the quantification range of the characterized control copy number. For quantification ranges for a specific assay, please contact Canon BioMedical. I ran technical replicates during a run and observed variation between them on the difference plots. Why is this? If the replicates do not cluster on the difference plots or have abnormal melt curves, it is usually caused by an insufficient amount in the PCR wells, not enough mixing, or sample contamination. Sometimes, well locations may also affect the melt curves. We recommend following good laboratory practice and testing the samples again. Contact Information We strive to deliver the highest quality technical support. Our support team is composed of experienced scientists with extensive academic and industry laboratory expertise in copy number analysis and PCR technologies. If you have any questions about our products or HRM, please do not hesitate to contact us. For technical assistance or more information, please visit our website at us at contactus@canon-biomedical.com, or call us at 844-CANONBIO ( ). Novallele Genotyping Assay User Guide Page 19 of 24

20 Ordering Information Product name Description Catalog number Novallele Copy Number Mastermix SMN1 Novallele Copy Number Assay SMN2 Novallele Copy Number Assay Rapid PCR chemistry formulation for copy number variation determination PCR assay to determine SMN1 copy number PCR assay to determine SMN2 copy number Page 20 of 24 Novallele Genotyping Assay User Guide

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23 Consider all registered names, trademarks, etc. used in this document, even when not specifically marked as such, as protected by law. Limited License Statements The purchase of this product grants the purchaser rights under certain Roche patents to use it solely for investigational research or providing human in vitro diagnostic services. No general patent or other license of any kind other than this specific right of use from purchase is granted hereby. Canon BioMedical warrants that each Novallele copy number assay and Novallele Copy Number Mastermix, each a Novallele Product, sold to the Customer will substantially perform in accordance with the written specifications therefor set forth in the product specification sheet originally included by Canon BioMedical with each Novallele Product delivered to the Customer until the instructed use by date set forth on the Novallele Product packaging. Canon BioMedical s sole liability for, and the Customer s sole remedy, under this limited warranty will be for Canon BioMedical to replace the nonconforming Novallele Product with a conforming Novallele Product, provided that in each case Canon BioMedical is notified and returned by the Customer during the warranty period of the nonconforming Novallele Product as instructed by an authorized Canon BioMedical employee. The warranty period on any replacement Novallele Product will be the period from the shipment date until the replacement Novallele Product use by date. A copy of the Canon BioMedical, Inc. standard terms and conditions applicable to the Novallele copy number assays and Novallele Copy Number Mastermix can be found on If you have questions about product specifications or performance, please call Canon BioMedical Support at 844-CANONBIO or visit Canon BioMedical, Inc. All Rights Reserved Rev. 01 Novallele Genotyping Assay User Guide Page 23 of 24

24 We are here to assist. Please or call to speak with our technical support staff about HRM or to place an order CANONBIO