Devyser AZF v2 RUO. Devyser AZF Extension RUO. Handbook

Transcription

1 Devyser AZF v2 RUO Art. No.: 8-A019.2-RUO Devyser AZF Extension RUO Art. No.: 8-A020-RUO For Research Use Only Handbook Page 1 of 34

2 Table Of Contents Table Of Contents 2 1. Introduction to Devyser AZF v2 RUO and Devyser AZF Extension RUO 4 Intended use 4 Included in the kits 6 Test procedure 6 Principle of the Procedure 6 2. Warnings and Precautions 7 3. Symbols used on Labels 9 4. Required Material Included in the Kits 10 Configurations 10 Components Devyser AZF v2 RUO 10 Components Devyser AZF Extension RUO Required but not Provided 10 Reagent Preparation 10 DNA Extraction 10 Amplification 10 Detection 11 Size Standard Dye Set Calibration Storage and Handling Requirements Sample Requirements 13 Clinical Samples 13 DNA Extraction and Measurement 13 Procedure and Storage 14 Controls Instructions for Use Workflow Devyser AZF v2 RUO and Devyser AZF Extension RUO Sample Preparation and PCR Amplification 15 DNA Extraction 15 Addition of Sample 16 Amplification Detection 17 Sample Preparation 17 Sample Preparation for Capillary Electrophoresis 17 Instrument Preparation 17 Run Modules Results and Analysis 19 Principle of Detection 19 Page 2 of 34

3 8.1 Data Interpretation 19 Data Analysis 19 Peak cutoff signals (RFU) 19 Negative Control 19 Normal Male Control 20 Normal Female Control 21 Clinical Samples 21 Markers sy254 and sy Marker sy84 23 Marker sy Marker Overview Devyser AZF v2 RUO 25 Marker Overview Devyser AZF Extension RUO 25 Troubleshooting 28 PCR Artefacts 28 Electrophoretic Artefacts Procedural Limitations Notice to Purchaser References Contact Information Revision History 34 Page 3 of 34

4 1. Introduction to Devyser AZF v2 RUO and Devyser AZF Extension RUO Intended use Devyser AZF v2 RUO For research use only, not for use in diagnostic procedures. Devyser AZF Extension RUO For research use only, not for use in diagnostic procedures. Workflow The workflow for the AZF screening with the Devyser AZF v2 RUO kit and the extension analysis with the Devyser AZF Extension RUO kit is described in the flow chart in Figure 1. Page 4 of 34

5 Figure 1. Flow chart of AZF screening and extension analysis Page 5 of 34

6 Included in the kits The Devyser AZF v2 RUO and the Devyser AZF Extension RUO kits contain ready-to-use reagents for PCR amplification of genetic markers. Test procedure DNA extraction: The Devyser AZF v2 RUO and Devyser AZF Extension RUO kits can be used with QIAamp DNA Blood Mini QIAcube Kit (Qiagen, cat.# 51126) and QIAsymphony DSP DNA Midi Kit (Qiagen, cat.# ) for extraction of DNA from whole blood (EDTA). Amplification: The Devyser AZF v2 RUO and Devyser AZF Extension RUO kits kits can be used with Life Technologies/Thermo Fisher Scientific Veriti Thermal cycler. Detection: Life Technologies/Thermo Fisher Scientific/ABI Genetic Analyzers (ABI 310, 3130, 3500, 3730) that support detection of Devyser Dye Set DEV-5. Principle of the Procedure Genetic testing using the Devyser AZF v2 RUO and Devyser AZF Extension RUO kits relies on PCR amplification of sequence-tagged sites (STS) in the AZFa, AZFb and AZFc regions of the Y-chromosome. Successful amplification of an STS marker indicates prescence whereas absence of PCR amplification is indicative of deletion. Primers for a total of 8 markers (Devyser AZF v2 RUO) and 13 markers (Devyser AZF Extension RUO) are combined into two separate multiplex PCR reactions. All the loci tested in the Devyser AZF v2 RUO and the Devyser Extension RUO kits are recommended by the European Academy of Andrology (EAA) and the European Quality Monitoring Network Group (EMQN) 1. The ZFXY marker is used as an internal control during PCR amplification, as the primers used amplify fragments from the Y-chromosome and the X-chromosome. The sy14 marker (SRY gene) is included in the analysis as a control for the testis determining factor on the short arm of Y chromosome and for the presence of Y-specific sequences in the case that the ZFY gene is absent. Page 6 of 34

7 2. Warnings and Precautions A. The Devyser AZF v2 RUO and Devyser AZF Extension RUO kits should be used with a total PCR reaction volume of 25 µl. Changing the reaction volume will compromise the kit performance. B. Avoid microbial contamination of reagents when removing aliquots from reagent vials. The use of sterile disposable aerosol barrier pipette tips is recommended. C. Do not pool reagents from different lots or from different vials of the same lot. D. Do not use a kit after its expiry date. E. Do not use opened or damaged kit reagent vials. F. Work flow in the laboratory should proceed in a unidirectional manner, beginning in the reagent preparation area and moving to the DNA extraction area and then to the amplification area and finally to the detection area. Pre-amplification activities should begin with reagent preparation and proceed to DNA extraction. Reagent preparation activities and DNA extraction activities should be performed in separate areas. Supplies and equipment should be dedicated to each activity and not used for other activities or moved between areas. Gloves should be worn in each area and should be changed before leaving that area. Equipment and supplies used for reagent preparation should not be used for DNA extraction activities or for pipetting or processing amplified DNA or other sources of target DNA. Amplification and detection supplies and equipment should remain in the amplification and detection area at all times. G. Handling of kit components and samples, their use, storage and disposal should be in accordance with the procedures defined by national biohazard safety guidelines or regulations. Page 7 of 34

8 H. Wear powder free disposable gloves, laboratory coats and eye protection when handling specimens and kit reagents. Wash hands thoroughly after handling specimens and kit reagents. Page 8 of 34

9 3. Symbols used on Labels Lot or batch number Expiry date Number of tests Store below temperature shown Catalogue number Manufacturer Page 9 of 34

10 4. Required Material 4.1 Included in the Kits Configurations The Devyser AZF v2 RUO and the Devyser AZF Extension RUO test kits contain reagents for analysis of 25 samples each. Components Devyser AZF v2 RUO Table 1. Components included in the Devyser AZF v2 RUO kit. Cap Colour Tube Colour Label Art.Nr. Kit content Orange Clear PCR Activator RUO 4-A035 1 White Amber AZF v2 Mix RUO 4-A241 1 Components Devyser AZF Extension RUO Table 2. Components included in the Devyser AZF Extension RUO kit. Cap Colour Tube Colour Label Art.Nr. Kit content Orange Clear PCR Activator RUO 4-A035 1 Yellow Amber Extension Mix RUO 4-A Required but not Provided Reagent Preparation Consumables for the Thermal Cycler Micropipette with aerosol barrier tips or dispenser with displacement tips Disposable protective gloves (powder free) DNA Extraction Reagents and equipment according to manufacturer s instructions for use Micropipette or multipipette with aerosol barrier tips Disposable protective gloves (powder free) Amplification Thermal Cycler: Life Technologies/Thermo Fisher Scientific Veriti Thermal cycler. For use of alternative thermal cyclers the following ramp rates must be applied: heating 1,6 C/s, cooling 1,6 C/s Micropipette or multipipette with aerosol barrier tips Disposable protective gloves (powder free) Page 10 of 34

11 Detection Life Technologies/Thermo Fisher Scientific/ABI Genetic Analyzer (ABI 310, 3130, 3500, 3730) Performance optimized polymers: POP-4 and POP-7 Hi-Di Formamide, Genetic Analysis Grade 1x Genetic Analyzer Buffer Micropipette or multipipette with aerosol barrier tips Disposable protective gloves (powder free) Size Standard 560 SIZER ORANGE (Devyser Art. No.: 8-A402) or GeneScan 600 LIZ Size Standard (Life Technologies cat.# ). 4.3 Dye Set Calibration ABI 3130, 3730: Use DEV-5 Dye Set MultiCap kit (Devyser Art. No.: 8-A401) in the Any5Dye Dye Set. ABI 3500: Use DEV-5 Dye Set MultiCap kit (Devyser Art. No.: 8-A401) and generate the DEV-5 Dye Set. ABI 310 Matrix file generation: Use DEV-5 Dye Set SingleCap kit (Devyser Art. No.: 8-A400). Run with module file GS STR POP4 (1 ml) G5.md5. Detailed instructions for Dye Set calibration may be downloaded from the download section at: Page 11 of 34

12 5. Storage and Handling Requirements A. Store all components below -18 C. B. The activated reaction mixes (prepared by addition of the AZF v2 Mix RUO and the Extension Mix RUO to separate tubes of PCR Activator) may be stored at +2 to +8 C for at least 7 days and below -18 C 12 months or until the kit expiration date. Avoid repeated freeze-thawing. C. Dispose of unused reagents and waste in accordance with country, federal, state and local regulations. D. Do not mix reagents from different kit lot numbers. Page 12 of 34

13 6. Sample Requirements Clinical Samples The Devyser AZF v2 RUO and the Devyser AZF Extension RUO kits can be used with human genomic DNA extracted from whole blood (EDTA) DNA Extraction and Measurement The Devyser AZF v2 RUO and the Devyser AZF Extension RUO kits can be used with QIAamp DNA Blood Mini QIAcube Kit (Qiagen, cat.# 51126) and QIAsymphony DSP DNA Midi Kit (Qiagen, cat.# ) for extraction of DNA from whole blood (EDTA). It is recommended that alternative DNA extraction methods and sample materials are thoroughly tested with the Devyser AZF v2 RUO and the Devyser AZF Extension RUO kits prior to use. For the recommended PCR conditions and analysis settings (see sections ), results are consistently obtained at DNA concentrations between 50 and 300 ng/pcr reaction (10-60 ng genomic DNA/µL sample). DNA concentration and DNA purity are important factors for successful testing using the Devyser AZF v2 RUO and the Devyser AZF Extension RUO kits. DNA should be free from contaminating protein, salts and other PCR inhibitors (e.g. residual ethanol from DNA extraction procedures). Poor quality DNA may result in amplification failure and increased background signals during detection. Accurate and reproducible quantitation of DNA concentration is important since addition of too much DNA to the PCR reaction can generate false positive results and addition of too little amount of DNA can cause amplification failure. High quality DNA is important for accurate and reproducible determination of DNA concentration in a sample. Due to variations in DNA quantitation techniques it is important that the user verifies that the DNA quantitation technique used for determination of DNA concentration correlates to the actual results obtained from the Devyser AZF v2 and the Devyser AZF Extension kits. The user should be aware of the variations and limitations of different methods and instruments available for DNA quantitation. All DNA concentrations referred to in this handbook were determined using Qubit dsdna HS Assay Kit (Life Technologies/Thermo Fisher Scientific, cat.# Q32851). The DNA concentration determined in a sample using Qubit dsdna HS Assay Kit may differ from the DNA concentration determined by other methods. Page 13 of 34

14 Procedure and Storage According to manufacturer s instructions for use. Controls It is recommended that suitable controls such as normal DNA and a negative control are included in each run. Page 14 of 34

15 7. Instructions for Use 7.1 Workflow Devyser AZF v2 RUO and Devyser AZF Extension RUO The activated reaction mixes should be prepared before preparing the samples, if the complete process is performed in one day. Only if the samples are prepared the day before amplification or earlier, the opposite order is advisable. The Devyser AZF v2 RUO and Devyser AZF Extension RUO should be used with a total PCR reaction volume of 25 µl. Changing the reaction volume will compromise the kit performance. The activated reaction mixes are prepared by adding the AZF v2 Mix RUO and the Extension Mix RUO to separate tubes of PCR Activator. Ensure that the AZF v2 Mix RUO and the Extension Mix RUO are completely thawed before use. 1. Centrifuge each tube briefly to collect the content. Do not vortex the tubes at this step. 2. Add 500 μl from the AZF v2 Mix RUO or the Extension Mix RUO to one tube of PCR Activator. 3. Carefully mix by pipetting several times from the bottom of the tube. 4. Vortex the activated reaction mix and centrifuge briefly to collect the content. 5. Add 20 µl of the activated reaction mix to separate PCR reaction tubes. 6. Cap the reaction tubes and centrifuge briefly to collect the contents. 7. Continue to step 7.2. The activated reaction mix is stable at +2-8 C for at least 7 days and below -18 C for 12 months or until the kit expiration date. Avoid repeated freeze-thawing. 7.2 Sample Preparation and PCR Amplification DNA Extraction According to manufacturer s instructions for use. It is recommended to use DNA concentrations between 50 and 300 ng/pcr reaction (10-60 ng genomic DNA/μL sample). Page 15 of 34

16 Addition of Sample Samples and controls should be added in a dedicated area separated from reagent preparation, amplification and detection areas. 1. Add 5 µl of clinical sample (10-60 ng genomic DNA/µL sample) to each PCR reaction tube containing activated reaction mix (from step 7.1). 2. Cap the tubes and centrifuge briefly to collect the content. Amplification The Devyser AZF v2 RUO and the Devyser AZF Extension RUO kits can be used with Life Technologies/Thermo Fisher Scientific Veriti Thermal cycler. PCR instruments should be regularly calibrated and maintained to ensure accurate PCR performance. For Life Technologies/Thermo Fisher Scientific Veriti Thermal cycler: In the "Tools Menu" select "Convert a Method". In the next step select "9700 Max Mode" and then enter the PCR profile as outlined below. Other thermal cyclers: For Life Technologies/Thermo Fisher Scientific/ABI GeneAmp PCR System 9700, set ramp speed to MAX. Important! On other thermal cyclers, set ramp rates to heating 1,6 C/s and cooling 1,6 C/s. Amplification Area: Program the Thermal Cycler for amplification according to the following thermal profile (consult the User's Manual for additional information on programming and operation of the thermal cycler): 1. Set reaction volume to 25 µl. 2. Start the amplification (duration approximately 2 hrs). 3. Following amplification, remove the tubes containing completed PCR amplification reaction from the thermal cycler and place into a suitable holder. Centrifuge briefly to collect the content. Remove the caps carefully to avoid aerosol contamination. Do not bring amplified material into the pre-amplification areas. Amplified material should be restricted to amplification and detection areas. Page 16 of 34

17 7.3 Detection Sample Preparation Refer to the respective Life Technologies/Thermo Fisher Scientific/ABI Genetic Analyzers User's Manual for instructions on maintenance and handling. Prior to running the Devyser AZF v2 RUO and the Devyser AZF Extension RUO kits, the instrument must be spectrally calibrated to support detection of the Dye Set DEV-5. See section 4.3 for details. Sample Preparation for Capillary Electrophoresis 1. Prepare a loading cocktail by combining and mixing 2 µl of the size standard (e.g. 560 SIZER ORANGE) with 100 µl Hi-Di Formamide (sufficient mix for 6 wells or tubes). 2. Vortex for 15 seconds. 3. Dispense 15 µl of the loading cocktail into the required number of wells of a microwell plate or into individual tubes (ABI 310) to be placed on the Genetic Analyzer. 4. Add 1,5 µl of the sample PCR product to the corresponding well or tube containing loading cocktail. 5. Seal the plate or the tubes. 6. Briefly centrifuge the plate or the tubes and ensure that the reagents do not contain any bubbles. 7. Load the plate or the tubes on the ABI Genetic Analyzer. Instrument Preparation Create a sample sheet using the data collection software with the following settings: Sample ID Dye Set: Any5Dye/DEV-5 Run module Run Modules The recommended run modules for different models of Life Technologies/Thermo Fisher Scientific Genetic Analyzers (ABI 310, 3130, 3500, 3730) are outlined in tables 2-5. The amount of PCR product injected into the capillaries can be adjusted by increasing/decreasing the injection time and/or injection voltage. Page 17 of 34

18 Table 2. Run module for ABI 310 (module file GS STR POP4 (1 ml) G5.md5 ). Run Parameters Capillary length Run temperature Injection voltage Injection time Run voltage Run time POP-4 47 cm 60 C 15 kv 5-15 s 15 kv 40 min Table 3. Run module for ABI Run Parameters Capillary length Run temperature Injection voltage Injection time Run voltage Run time POP-4/POP-7 36 cm 60 C 1,5 kv 20 s 15 kv 1500 s Table 4. Run module for ABI Run Parameters Capillary length Run temperature Injection voltage Injection time Run voltage Run time POP-7 50 cm 60 C 1,6 kv 15 s 19,5 kv 1500 s Table 5. Run module for ABI Run Parameters Capillary length Run temperature Injection voltage Injection time Run voltage Run time POP-7 36 cm 60 C 1,6 kv 15 s 15 kv 1500 s Page 18 of 34

19 8. Results and Analysis Principle of Detection Chromosome-specific markers known as sequence tagged sites (STS) are amplified by PCR. By the use of fluorescently labeled primers the visualization and identification of the PCR products are performed using a Genetic Analyzer with associated software. 8.1 Data Interpretation Data Analysis The marker peaks in the electrophoretogram should be identified according to the specific marker sizes as presented in the marker overview (Table 6 for Devyser AZF v2 and Table 7 for Devyser AZF Extension). Presence of an STS marker fragment is consistent with a non-deleted STS marker. Absence of an STS marker is consistent with a deleted STS marker. Peak cutoff signals (RFU) It is recommended that an instrument specific cutoff range is determined. Do not use lower cutoff values than 200 RFU (ABI 310, 3130) and 500 RFU (ABI 3730, 3500). Confirm that the control samples are approved prior to analyzing the clinical samples. Confirm that the control markers ZFXY and sy14 are present and above cutoff prior to performing the analysis of a clinical sample. Negative Control No specific peaks should be present within the range of bp (Devyser AZF v2 RUO) and bp (Devyser AZF Extension RUO). Page 19 of 34

20 Normal Male Control The number and length of the expected peaks are indicated in the marker overview (Tables 7 and 8). All peaks according to the marker overview must be present. See Figure 2a (Devyser AZF v2 RUO) and Figure 2b (Devyser AZF Extension RUO). Figure 2a. Typical results obtained from a normal male sample with Devyser AZF v2 RUO. All STS markers are present. Figure 2b. Typical results obtained from a normal male sample with Devyser AZF Extension RUO. All STS markers are present. Page 20 of 34

21 Normal Female Control Only one peak generated from marker ZFXY should be present. See Figure 3a (Devyser AZF v2 RUO) and Figure 3b (Devyser AZF Extension RUO). Figure 3a. Typical results for obtained from a normal female sample with Devyser AZF v2 RUO. All Y-chromosomal STS markers are absent and only the ZFXY marker is detected. Figure 3b. Typical results for obtained from a normal female sample with Devyser AZF Extension RUO. All Y-chromosomal STS markers are absent and only the ZFXY marker is detected. Clinical Samples Determine the presence or absence of marker specific peaks according to the flow chart in Figure 1 and tables All deletions should be contiguous. If a single marker peak or multiple Page 21 of 34

22 marker peaks that do not map to adjacent regions of the Y chromosome are missing, they may represent dropout peaks and may not be indicative of deletions. Consult the EAA/EMQN best practice guidelines 1 for additional information. Results from an abnormal sample (NA18340, Coriell Cell Repositories) with a complete AZFc deletion are shown in Figure 4a (Devyser AZF v2 RUO) and Figure 4b (Devyser AZF Extension RUO). Figure 4a. Typical results obtained from an abnormal male sample with Devyser AZF v2 RUO. Markers sy255 and sy254 are absent which is consistent with deletion of the AZFc region. Figure 4b. Typical results obtained from an abnormal male sample with Devyser AZF Extension RUO. Markers sy1192, sy153, sy1191 and sy1291 are absent which is consistent with complete deletion of the AZFc region (b2/b4). Page 22 of 34

23 Markers sy254 and sy255 STS markers sy254 and sy255 are present in at least 4 copies each on the Y-chromosome. In male samples with intact STS loci, both markers may generate peak heights exceeding the peak height of all other markers detected. Marker sy84 A single-nucleotide polymorphism (SNP), rs , has been reported in the primer binding site of the sy84 marker 2. The Devyser AZF v2 RUO kit has been tested with a sample having this SNP in the sy84 marker. During AZF screening with Devyser AZF v2, this SNP may result in a reduced sy84 marker signal as compared to other markers (see figure 5). In samples with low DNA concentration (below 10 ng/µl) or samples with poor DNA quality, this SNP may however cause an allele dropout of the sy84 marker. In these cases, it is advisable to rerun the sample with freshly extracted DNA or to perform sequencing of the sy84 primer binding sites to eliminate the possibility of a false negative result for the sy84 marker. Figure 5. Typical Devyser AZF v2 RUO results from a sample with a SNP (rs ) in the sy84 marker. This sample also has an AZFc deletion. The sample DNA concentration is 19 ng/µl. Page 23 of 34

24 Marker sy1291 Marker sy1291 contains a poly-t/a stretch which may cause marker size shifts within the indicated interval (grey bin in Figure 6). This is due to normal variations between individuals. Multiple stutter peaks are present in the sy1291 marker (see Figure 6). For interpretation of the gr/gr deletion, i.e. absence of marker sy1291 in Devyser AZF Extension, please consult the EAA/EMQN best practice guidelines 1 for additional information. Figure 6. Typical Devyser AZF v2 RUO results for the sy1291 marker with multiple stutter peaks. Page 24 of 34

25 Marker Overview Devyser AZF v2 RUO Table 6. Marker overview Devyser AZF v2 RUO STS Marker Peak colour Map position (Table 8) Marker size (±2,5 bp)** sy255* Blue sy127* Blue sy134* Blue sy86* Blue sy84* Blue sy254* Blue ZFXY Blue sy14 Blue Marker Overview Devyser AZF Extension RUO Table 7. Marker overview Devyser AZF Extension RUO STS Marker** Peak colour Map position (Table 8) Marker size (±2,5 bp)** sy160 Blue sy1191 Blue ZFXY Blue sy14 Blue sy1291 Blue sy88 Green sy1065 Green sy82 Green sy83 Green sy153 Yellow sy121 Yellow sy1192 Yellow sy105 Yellow *Basic set of STS primers according to EAA/EMQN best practice guidelines 1. **Extended set of STS primers according to EAA/EMQN best practice guidelines 1. ***Allele fragment lengths given in this table are based on average observed fragment lengths obtained using the POP-7 polymer (ABI3500) and POP4 polymer (ABI310), and 560 SIZER ORANGE. Allele fragment size ranges may vary depending on the instrument, polymer type and size marker used during electrophoresis. Page 25 of 34

26 Table 8. Schematic overview of the marker positions on the Y-chromosome. *AZF regions (AZFa, AZFb and AZFc) according to the EAA/EMQN best practice guidelines 1 and chromosome positions according to the UCSC Genome Bioinformatics Site 2. Page 26 of 34

27 Table 9. Predicted STS marker pattern* for AZFa, AZFb and AZFc deletions when performing AZF screening with Devyser AZF v2 RUO. AZFa deletion AZFb deletion AZFc deletion sy14 ZFXY sy86 sy84 sy127 sy134 sy254 sy Table 10. Predicted STS marker pattern* for AZFa, AZFb, AZFbc, gr/gr and AZFc deletions when performing extension analysis with Devyser AZF Extension RUO. AZFa deletion AZFb deletion AZFbc deletion gr/gr deletion AZFc deletion (b2/b4) AZFc deletion (terminal) sy14 ZFXY sy82 sy83 sy1065 sy88 sy105 sy121 sy1192 sy153 sy1191 sy1291 sy *Other STS marker patterns may appear. In these cases, consult the EAA/EMQN best practice guidelines 1 and the troubleshooting section and for additional information. Page 27 of 34

28 Troubleshooting In the case of the absence of a single STS marker (not applicable for gr/gr) or multiple markers that do not map to adjacent regions, it is recommended that follow-up studies are performed to identify the reason. Consult the EAA/EMQN best practice guidelines 1 for additional information. If electrophoretograms are of poor quality the data should not be interpreted. The PCR product may be re-injected and re-analyzed. In case of inconclusive results or a if single marker is not detected, a number of explanations are possible:» Primer site deletion» Primer site polymorphism/mutation.» Crosstalk between dye-channels» Electrophoretic spike» Contaminating DNA: second genotype, PCR amplicons» DNA concentration is too high or too low» DNA is degraded or of poor quality PCR Artefacts -A peaks (figure 5) are detected as extra peaks that is one base pair shorter than the full length (+A peak) PCR product. Figure 5. -A and +A peaks as indicated by the arrows. Page 28 of 34

29 Electrophoretic Artefacts Crosstalk/bleed through between dye channels may occur during detection (Figure 6). Crosstalk appears as equally sized peaks in neighbouring dye channels and should be excluded from the analysis. Figure 6. Crosstalk peak (from green to blue channel) as indicated by the arrow. Dye blobs may appear in the sample analysis range (Figure 7). In general, dye blobs appear as broad, undefined peaks of a single colour and tend to occur relatively early in the data. Figure 7. Dye blob as indicated by the arrow. Page 29 of 34

30 9. Procedural Limitations A. This product is for research use only. Performance characteristics have not been established. Page 30 of 34

31 10. Notice to Purchaser Results from Devyser AZF v2 RUO and AZF Extension RUO should not be used for diagnostic purposes. These products are intended for research use only. LIZ, Veriti and GeneAmp are registered trademarks of Life Technologies/Thermo Fisher Scientific Corporation. GeneScan, POP-4, POP-7 and Hi-Di are trademarks of Life Technologies/Thermo Fisher Scientific Corporation. Purchase of this product does not provide a license to perform PCR under patents owned by any third party. Page 31 of 34

32 11. References 1 C. Krausz, L. Hoefsloot, M. Simoni and F. Tuttelman. Andrology, 2014, 2, EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdelitions: state-of-the-art Wu Q, Chen G, Yan T, Wang H, Liu Y, Li Z, Duan S, Sun F, Feng Y and Shi H. Prevalent false positives of azoospermia factor a (AZFa) microdeletions caused by single-nucleotide polymorphism rs in the sy84 screening of male infertility. Asian J Androl Nov; 13(6): The UCSC Genome Bioinformatics Site, Cited Page 32 of 34

33 12. Contact Information Devyser AB Instrumentvägen 19 SE Hägersten SWEDEN Phone: Homepage: Technical Support Phone: Page 33 of 34

34 13. Revision History Table 9. Updates in Devyser AZF v2 RUO as compared to Devyser AZF RUO (version 1) STS Marker Locus Marker size (±2,5 bp) Change in v2 sy131 DYS bp Excluded sy90 DYS bp Excluded sy81 DYS bp Excluded sy625 G bp Excluded sy157 DYS bp Excluded M259 DDX3Y 396 bp Excluded ZFY/ZFX* ZFY / ZFX 432/433 bp* Fragment length, name sy14 (SRY)* SRY 464 bp Name *See table 6 for fragment lengths and names in the Devyser AZF v2 kit. Page 34 of 34