Y CHROMOSOME MICRODELETION Detection System v.4.0

Transcription

1 Y CHROMOSOME MICRODELETION Detection System v.4.0 India Contact: Life Technologies (India) Pvt. Ltd. 306, Aggarwal City Mall, Opposite M2K Pitampura, Delhi (INDIA). Ph: , , Mobile: Fax: Web:

2 Contents Section 1: Introduction... 1 Section 2: Product Overview... 2 Description of Test... 2 Chromosomal regions of STS... 4 Performing the Test... 5 Laboratory Design and Organization... 5 Workflow Overview... 6 Section 3: Materials, Safety Warnings and Precautions... 7 Kit Contents... 7 Safety Warnings and Precautions... 8 Shipping Conditions... 9 Storage and Handling Requirements... 9 Section 4: Y Chromosome Microdeletion Detection Kit Procedure Preparing Genomic DNA Template Preparing PCR Section 5: Electrophoretic Detection and Data Analysis Performing Capillary Electrophoresis with an ABI Genetic Analyzer Data Analysis with GeneMapper Software v Preparing the Software for Analysis External Control Patterns for All Panels Section 6: Troubleshooting Assignment of Size Standard Validation of the Y Chromosome Microdeletion Detection Kit Y CHROMOSOME MICRODELETION DETECTION İ

3 Section 1: Introduction Microdeletions of the Y chromosome are the second most frequent genetic cause of spermatogenetic failure in infertile men after Klinefelter syndrome. The molecular diagnosis of Y chromosomal microdeletions is routinely performed in the workup of male infertility in men with azoospermia or severe oligozoospermia (M. Simoni et al., 2004). The portion of the male-specific region of the Y chromosome (MSY), comprising 95% of the Y chromosome and flanked by pseudoautosomal regions (Skaletsky et al., 2003), which is affected by deletions, has been classically subdivided into four regions called AZFa, AZFb, AZFc and proximal AZFc (AZFd), respectively. Microdeletions are relatively frequent among infertile men, although the incidence can vary considerably depending on the selection criteria of the patients. Azoospermic men have a higher incidence of microdeletions than oligozoospermic men and consequently the deletion frequency found in different laboratories may vary from 2 to 55% (or even higher), reflecting the composition of the study population. The AZFa region is about 1100 kb long and contains the single copy genes DFFRY (or USP9Y) and DBY. In any case the complete deletion of the AZFa region removes about 792 kb including both USP9Y and DBY genes, the only two genes in AZFa. Deletions involving only the USPY9 gene or the DBY gene have been reported only by one group (Ferlin et al., 1999; Foresta et al., 2000). The type and mechanism of deletions of the AZFb and AZFc region have been recently clarified as well (Kuroda- Kawaguchi et al., 2001). Both regions together comprise 24 genes, most of which present in multiple copies for a total of 46 copies. The complete deletion of AZFb removes 6.2 Mb (including 32 copies of genes and transcription units) and results from homologous recombination between the palindromes P5/proximal P1 (Repping et al., 2002). The AZFc region includes 12 genes and transcription units, each present in a variable number of copies making a total of 32 copies (Repping et al., 2003). The classical complete deletion of AZFc, the most frequent pattern among men with deletions of the Y chromosome, removes 3.5 Mb, originates from the homologous recombination between amplicons b2 and b4 in palindromes P3 and P1 respectively and removes 21 copies of genes and transcription units (Kuroda-Kawaguchi et al., 2001). 1

4 Section 2: Product Overview Description of Test This system consists of 14 primer pairs that are homologous to previously identified and mapped sequence-tagged sites (STS). Y chromosome deletions in the regions that are amplified by these primer sets have been associated with male infertility. This system uses a five-dye fluorescent system for automated DNA fragment analysis, which allows multiplex amplification and electrophoresis of over 14 STS which include AMXY marker (Chr.X 104bp, Chr.Y 109bp; Xp22.1, Yp11.2) simultaneously. The kit is intended for use on Applied Biosystems ABI PRISM genetic analysis instrumentation. Fluorochromes include FAM, JOE, ATTO550 and ATTO565, to be used in conjunction with GeneScan 500 LIZ size standard (Applied Biosystems PN ). The Y-Del Primer Mix includes a primer pair to amplify chromose-specific sequences of the paralogous gene Amelogenin, which has a high degree of sequence identity between chromosomes Chr.X and Chr.Y. However nucleotide differences occur within each locus and can be used to generate chromosespecific PCR product. The primer pair included in the Y-Del Primer Mix exploits a 5bp deletion (Chr.X 104bp, Chr.Y 109bp; Xp22.1, Yp11.2) to generate one Chr.X-specific product that is 5bp shorter than the corresponding product on Y chromosome. Although the Y Chromosome Microdeletion Detection Kit has been designed for the detection of microdeletions occurring on the q-arm of the Y chromosome, relative quantification between Chr.X and Chr.Y allows assessment of Klinefelter syndrome, the most significant factor in male infertility. However, comparative assessment of the AMXY marker cannot be used for the standalone diagnosis of Klinefelter syndrome. Further investigations are necessary for precise diagnosis. Control primer pairs are internal controls for the amplification reaction and the integrity of the genomic DNA sample. The Primer Mix includes a primer pair that amplifies a region of the SRY gene. This is a control for the testis determining factor on the short arm of the Y chromosome and allows detection of XX males arising from Y to X translocations. The Y Chromosome Microdeletion Detection Kit has been extensively tested for reproducibility and robustness, and quality controlled to ensure optimum performance. The system incorporates controls to ensure performance of the Y CHROMOSOME MICRODELETION DETECTION 2

5 amplification reagents and to identify problems with genomic DNA samples. A positive control Male Genomic DNA is included for use in evaluating the amplification reactions. The presence of the appropriate amplification products in the positive control reactions indicates that the reagents are performing as expected. With the GML Y Chromosome Microdeletion Detection Kit, 14 markers are performed successfully. Of those, 6 markers (Sy84, Sy86, Sy127, Sy134, Sy254, and Sy255) are determined by European Molecular Genetics Quality Network guidelines. The remaining 6 are markers for all regions and so verify the Detection Kit in addition to control markers (SRY and AMXY). STS included in the Y Chromosome Microdeletion Detection Kit Marker Region bp Dye SRY(Y14) Control 464 JOE Sy255 AZFc 123 Sy134 AZFb 303 JOE JOE AMXY Control Chr.X 104 Chr.Y 109 FAM RBMY RBMY 238 FAM AZFa AZFa(Prox2) 217 FAM Sy133 AZFb 176 FAM Sy153 AZFd 136 FAM Sy152 AZFd 124 ATTO565 Sy157 AZFc 291 ATTO565 Sy84 AZFa 330 ATTO565 Sy254 AZFc 382 ATTO565 Sy86 AZFa 317 ATTO550 Sy127 AZFb 271 ATTO550 Fragment sizes may vary up to 3 bp depending on the instrument and electrophoresis conditions employed. Sizes in this table have been obtained on an ABI 3130xL Genetic Analyser using the 36cm capillary array, POP7 polymer, FragmentAnalysis_36_POP7_1 default module and G5 Dye Set. Y CHROMOSOME MICRODELETION DETECTION 3

6 Chromosomal regions of STS Y CHROMOSOME MICRODELETION DETECTION 4

7 Performing the Test The Y Chromosome Microdeletion Detection Kit contains all necessary reagents for the amplification of human genomic DNA. The reagents are designed for use with the following Applied Biosystems instruments: ABI PRISM 3100/3100-Avant Genetic Analyzer Applied Biosystems 3130/3130xl Genetic Analyzer Applied Biosystems 3500/3500xl Genetic Analyzer Applied Biosystems 310 Genetic Analyzer GeneAmp PCR System 9600 or 9700 Laboratory Design and Organization Special consideration should be given to the design and organization of the laboratory. The laboratory must be organized so that the area in which amplified DNA is handled is physically isolated from the work areas for DNA extraction and PCR setup. Strict physical isolation must be maintained between the area designated for handling amplified DNA and the other areas, to avoid transfer of amplified DNA out of the designated work area. Amplified DNA or equipment and supplies used to handle amplified DNA should not be taken out of the designated work area. If the work area for amplified DNA is in a separate but contiguous room, make sure that air flows toward the amplified DNA area. In addition, it is helpful if there is a separate exit from the amplified DNA work area that does not exit into the pre-pcr work areas. The laboratory should have four designated Work Areas, each ideally equipped with dedicated equipment and supplies to minimize the potential for contamination: 1. Evidence Handling Work Area: Pieces of evidence are examined, photographed, and divided for analysis. 2. DNA Extraction Work Area: This work space is used to perform the extraction steps. 3. PCR Setup Work Area: PCR reagent and DNA sample additions are made here. 4. Amplified DNA Work Area: This area is dedicated to PCR amplification and detection, and other activities that require handling of amplified DNA. Y CHROMOSOME MICRODELETION DETECTION 5

8 Workflow Overview Extract DNA Quantify DNA Perform PCR Perform Electrophoresis Analyze Data Y CHROMOSOME MICRODELETION DETECTION 6

9 Section 3: Materials, Safety Warnings and Precautions Kit Contents Component Description 25 X Volume Storage Y-DEL PCR Mix Contains salts, dntps and glycerol. 300 µl -20 o C GML Taq. Pol. Contains enzyme (5U/µL). 15 µl -20 o C Y-DEL Primer Mix Contains forward and reverse primers to amplify human Y-Chromosome targets. 300 µl -20 o C Control DNA Contains 10 ng/µl human male DNA. 30 µl -20 o C Y CHROMOSOME MICRODELETION DETECTION 7

10 Safety Warnings and Precautions Please be sure to observe the following warnings. Warning 1. For research use only. 2. Not for diagnostic purpose. 3. Use appropriate laboratory practices for sample handling. 4. Wear protective disposable gloves and eye protection when handling diluents and human DNA. 5. Wash hands properly after handling diluents and human DNA. 6. Use recommended materials, procedures, and equipment only. 7. Do not eat, drink or smoke in work areas. 8. Use sterile disposable pipettes and filtered pipette tips. 9. Do not mix reagents from different lots. 10. Do not use more or less than the required volumes of reagents. 11. Do not use any expired reagents. 12. To reduce the risk of contamination, the area where amplified DNA is handled must be physically isolated from the work areas for sample preparation and PCR setup. 13. Store the fluorescent dye labeled reagents in the dark <-15 o C. 14. Open and close all sample tubes or wells carefully to avoid splashing or spraying. Caution 15. Minimize the exposure of Hi-Di Formamide to the air and do not contaminate Hi-Di Formamide when sampling. 16. Chemical Hazard. Pop-7 polymer causes eye, skin, and respiratory tract irritation. It is a possible reproductive and birth defect hazard. Please read the Applied Biosystems MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. 17. Chemical Hazard. Formamide causes eye, skin, and respiratory tract irritation. It is a possible reproductive and birth defect hazard. Please read the Applied Biosystems MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Y CHROMOSOME MICRODELETION DETECTION 8

11 Shipping Conditions The GML Y CHROMOSOME MICRODELETION DETECTION KIT is shipped on ice packs and should still be frozen on arrival. Avoid unnecessary freeze-thawing of the contents of the kit. Storage and Handling Requirements Fluorescent primers should be stored away from light. The GML Y Chromosome Microdeletion Detection Kit box is internally coated to increase light protection. This kit is stable for up to one year if stored at -20 ºC. PCR and primer mixes can be stored as ready to use aliquots in PCR tubes at -20 ºC; this will keep freeze-thaw cycles to a minimum reducing the risk of contamination and shortening PCR set-up times. Y CHROMOSOME MICRODELETION DETECTION 9

12 Section 4: Y Chromosome Microdeletion Detection Kit Procedure Preparing Genomic DNA Template 1. Prepare genomic DNA according to standard protocols. 2. Determine the concentration by A260 or fluorescence. 3. The amount of DNA required is ng/µl. Preparing PCR The Y Chromosome Microdeletion Detection Kit has been optimized for use with the Perkin-Elmer GeneAmp PCR System 9600 or Prepare 1 PCR tube for each sample and external control. 2. Thaw vials to be used and mix thoroughly by vortexing for a few minutes. 3. Aliquot PCR Mix into each PCR tube as shown in the table below. Component Volume PCR Mix 10.0 µl Primer Mix 10.0 µl Taq. Pol. 0.5 µl Total 20.5 µl 4. Add 5.0 µl of DNA (10 50 ng/µl) to each PCR tube. Starting the Reaction Transfer PCR tubes to a PCR machine and begin thermocycling with the following program: Stage Description Temp. Time 1 Activation 95 ºC 10 min. Amplification 95 ºC 45 sec cycles 58 ºC 1 min. 72 ºC 1 min. 3 Final Extension 72 ºC 7 min. 4 Hold 4 ºC Indefinite Hold Note: For the GeneAmp PCR System 9600, the heat/cool rate is 2.3 o C per second whereas it is 5 o C per second for the When the GeneAmp PCR System 9600 is used, the peak heights will be too high, making analysis difficult, therefore the injection time should be decreased to a minor value (min/sec) to display normalized peaks when the samples are loaded onto the Genetic Analyzer (See Sec.6). Y CHROMOSOME MICRODELETION DETECTION 10

13 Section 5: Electrophoretic Detection and Data Analysis Performing Capillary Electrophoresis with an ABI Genetic Analyzer 1. Prepare the necessary amount of size standard for all samples to be analysed by combining: To run samples on 3100, 3100 Avant, 3130xL, 3730xL, 3500xL Genetic Analyzers: 10µl Hi-Di Formamide 0.3 µl GeneScan -500 LIZ To run samples on the 310 Genetic Analyzer: 20 Hi-Di Formamide 0.5 µl GeneScan -500 LIZ 2. For 3100, 3100 Avant, 3130xL, 3730xL, 3500xL Genetic Analyzers: Use 10 µl of this mix to inject 1 µl of PCR products collected in the same tube. For the 310 Genetic Analyzer: Use 20 µl of this mix to inject 1.5 µl of PCR products collected in the same tube. 3. Create Plate Sample Sheet using the Data Collection Software: Select the appropriate Result Group. Select the appropriate Instrument Protocol for 3100, 3130 and 3130xL, 3500xL instruments, or the appropriate Run Module for 310, using the following specifications: Instrument Type Run Modules and Conditions References 3130/3130xL 3500xL Analyzer 3100 Analyzer 310 Analyzer FragmentAnalysis36_POP7_1, Dye Set G5 FragmentAnalysis36_POP7_1, Dye Set G5 GS STR POP4 (1mL) G5 v2.md5 Injection condition: 15 kv/5 sec ABI PRISM 3130/ 3130xl, 3500xL Genetic Analyzer User s Manual ABI PRISM 3100/3100- Avant Genetic Analyzer User s Manual ABI PRISM 310 Genetic Analyzer User s Manual Y CHROMOSOME MICRODELETION DETECTION 11

14 Data Analysis with GeneMapper Software v5.0 Preparing the Software for Analysis Double click to open GeneMapper software on desktop. Login with User Name and Password to access the software. Y CHROMOSOME MICRODELETION DETECTION 12

15 To import the panels from the CD provided with the kit, select Tools > Panel Manager. Select Panel Manager. Y CHROMOSOME MICRODELETION DETECTION 13

16 Click File > Import Panels. Select GML_YdEL_Panels and click Import. Y CHROMOSOME MICRODELETION DETECTION 14

17 To import the binsets from the CD provided with the kit, click on GML_YdEL. Click File > Import Bin Set. Y CHROMOSOME MICRODELETION DETECTION 15

18 Select GML_YdEL_bins and click Import. Click Apply and OK. To import the other parameters from the CD provided with the kit, select Tools > GeneMapper Manager. Y CHROMOSOME MICRODELETION DETECTION 16

19 On the Analysis Methods tab, click Import. Choose GML_Ydel_anl file and click Import. 1 2 GML_ CVD-1 Panel_anlys file for Analysis GML_ CVD-1 Panel_rep file for Report GML_ CVD-1 Panel_size file for Size GML_ CVD-1 Panel_tbl file for Table Settings GML_ CVD-1 Panel_plt file for Plot Settings 3 Methods Settings Standard Import the other files below in the same manner: On Table Settings tab: GML_YdEL_tbl On Plot Settings tab: GML_YdEL_plt On Report Settings tab: GML_YdEL_rep On Size Standard tab: GML_YdEL_size Click Done. Y CHROMOSOME MICRODELETION DETECTION 17

20 To open a New Project, click on the icon and choose Microsatellite Click OK. Click on the icon, to Add Samples to Project. Choose the pathway for the run files, click Add to List and Add. More samples can be selected with CTRL button on keyboard Y CHROMOSOME MICRODELETION DETECTION 18

21 To change the Table Settings, open Table Settings tab and select GML_YdEL_tbl in the drop-down list. Select GML_YdEL in the drop-down lists under Analysis Method, Panel and Size Standard as indicated below. Analysis Method: Select GML_YdEL_anl Y CHROMOSOME MICRODELETION DETECTION 19

22 Panel: Select GML_YdEL Size Standard: Select GML_YDEL_size Select all of the boxes by holding left mouse button and pressing CTRL+D buttons on keyboard. Click on the Analyze icon. Y CHROMOSOME MICRODELETION DETECTION 20

23 Define a Name for the project created. Analysis begins. Y CHROMOSOME MICRODELETION DETECTION 21

24 Before analyzing the samples, ensure that the size peaks are correctly named as 75, 100, 139, 150, 160, 200, 300, 340, 350, 400, 450, 490 and 500 respectively (for more detailed definitions, see Sec. 6). Select the Samples tab. Select samples to view by pressing CTRL button and click plots. to display Y CHROMOSOME MICRODELETION DETECTION 22

25 External Control Patterns for All Panels Positive male genomic DNA (10 ng/µl) is included in the external control and the patterns for all markers are shown below. Before analyzing the samples, check all markers and bins for external control, and then evaluate the samples. Y CHROMOSOME MICRODELETION DETECTION 23

26 Y CHROMOSOME MICRODELETION DETECTION 24

27 Section 6: Troubleshooting This section covers problems and difficulties which may be encountered while using the kit, and their solutions. Assignment of Size Standard After analyzing the samples added to GeneMapper, the Size Match Editor should be carefully checked to confirm the sizing data of the samples (even if all of the samples have green sizing quality), because the correct assignment of peaks is a prerequisite for accurate analysis. If the Sizing Quality (SQ) flag is, it means that the samples pass the peak quality value. Note that peak assignments should be visually checked even if the SQ flag is green. If the Sizing Quality (SQ) flag is or, select (Size Match Editor) to view the size standard and peak assignments. If the peak assignments are correct, override the size quality value by clicking the button. If the peak assignments are incorrect for one or more samples, define a new size standard for the affected sample(s) as described above and reanalyze the sample(s). Y CHROMOSOME MICRODELETION DETECTION 25

28 Intensive Peaks As previously mentioned, fragments may be too intensive depending upon the thermal ramp value of the PCR machine. The intensity of fragments causes reflection to other dyes. The GML Y Chromosome Microdeletion Kit is optimized for maximum PCR product with 2.3 o C/sec ramp rate. In this case, the injection time must be decreased for accurate analysis. In the tree pane of the Data Collection software, click GA Instruments > Module Manager to open the Module Manager window. Click New and Run Module Editor dialog box opens. Complete the Protocol Editor dialog box as follows: Select FragmentAnalysis36_POP7 in the drop-down list. Select Regular in the Type drop-down list. Decrease Injection_Time from 16 sec to 12 sec. Type the name of the run module as GML_YdEL and click OK. Y CHROMOSOME MICRODELETION DETECTION 26

29 In the tree pane of the Data Collection software, click GA Instruments > Protocol Manager to open the Protocol Manager window. In the Instrument Protocols pane, click New and the Protocol Editor dialog box opens. Complete the Protocol Editor dialog box as following: Type the name as GML_YdeL. Select Regular in the Type drop-down list. Select GML_YdeL in the Run Module drop-down list. Select GML-5DYE as Dye Set. Click OK. Y CHROMOSOME MICRODELETION DETECTION 27

30 Validation of the Y Chromosome Microdeletion Detection Kit Different Concentrations of DNA The GML Y Chromosome Microdeletion Detection Kit has been studied and validated on numerous templates, indicating that the GML Y Chromosome Microdeletion Detection Kit responds to various concentrations of DNA. The GML Y Chromosome Microdeletion Detection Kit has also been validated with amnion DNA extracted by Chelex. 5 ng/µl * 10 ng/µl 20 ng/µl Y CHROMOSOME MICRODELETION DETECTION 28

31 Excessive Concentration of DNA If the concentration of DNA is decreased below 10 ng/µl, the peak heights are lowered, which complicates analysis. However too much concentrated DNA causes contamination due to the reflection of other dyes. Some extra peaks can be seen and pink reflection images can be an obstacle to evaluating the usual peaks. Furthermore, extensive peaks pick up other dyes of the same size. The GML Y Chromosome Microdeletion Detection Kit is optimized for ng/µl DNA. 100 ng/µl Y CHROMOSOME MICRODELETION DETECTION 29

32 Marker Amplification of Amelogenin Gene With female DNA only the single copy of amelogenin gene located on the X chromosome can be amplified as indicated below. With male DNA, AMELX is amplified along with AMELY due to the existence of the amelogenin gene on both chromosomes. Male Female AMELX s intron 1 contains a 6 bp deletion relative to intron 1 of AMELY. Y CHROMOSOME MICRODELETION DETECTION 30

33 Sample DNAs with Microdeletions on Y Chromosome Sample DNA (40 ng/µl) with multiple deleted markers Y152, Y153, Y133, Y134, RBMY, Y255, Y127, Y157, and Y254 is demonstrated in the table below. *Applied Biosystems, GeneScan, Genetic Analyser, ABI, Hi-Di, GeneMapper are products of Thermo Fisher Scientific, Inc. Y CHROMOSOME MICRODELETION DETECTION 31

34 Sample DNA (45 ng/µl) with multiple deleted markers Y153, Y255, Y152, Y157, and Y254 is demonstrated in the table below. Life Technologies (India) Pvt. Ltd. 306, Aggarwal City Mall, Road No. 44, Pitampura, Delhi , India Mobile: , Tel: , 8111, 8222, Fax: ; Y CHROMOSOME MICRODELETION DETECTION 32