Operating Instructions

Transcription

1 Sequazyme DNA Standards Kit Operating Instructions 1 Product Description The Sequazyme DNA Standards Kit allows you to do either or both of the following: Test the MassGenotyping Solution 1 (MGS1) System, excluding the SymBiot XT Workstation Check and optimize the resolution and sensitivity of the Voyager Biospectrometry Workstation (linear mode), used alone or as part of the MGS1 System The kit contains a mixture of three oligonucleotides that simulate the products of a primer extension reaction run using the Sequazyme Pinpoint SNP Assay Kit. The three oligonucleotides produce a mass spectrum that simulates a primer and its A/T heterozygote extensions. Figure 1 illustrates how to use the DNA Standards Kit. Apply Matrix and Primer Extension Mixture to a Voyager Sample Plate Test the MGS1 System Prepare the THAP Matrix Prepare the Primer Extension Mixture Check and Optimize Voyager Workstation Resolution and Sensitivity 3 Materials Materials Provided The DNA Standards Kit contains: THAP Matrix 2, 4, 6 trihydroxyacetophenone, 1 vial (40 mg) Matrix Diluent 2 vials (1.5 ml each) Primer Extension Mixture 1 vial (25 µg), containing: Oligonucleotide Materials Not Provided To use this kit, you need a: Sequence (5 to 3 ) Concentration When Dissolved in 25 µl Water Fresh 200-µL MicroAmp reaction tube with cap Voyager sample plate Mass (Da) R CTT TGT TCT GGG TTT C-3 2 µm 4,860 R377-17A 5 -CTT TGT TCT GGA TTT CA-3 1 µm 5,156 R377-17T 5 -CTT TGT TCT GGA TTT CT-3 1 µm 5,147 NOTE: If you use this kit to test the MGS1 System, do not use a 96 2-position, flat, hydrophobic plastic surface plate. Use only a sample plate supported by the MGS1 software, such as: 96-position, barcoded 100-position 384-position 384-position, barcoded 400-position Figure 1 Using the DNA Standards Kit Contents Page 2 Instrument Safety Before using the Voyager Workstation, read the Safety and Compliance section in the Voyager Biospectrometry Workstation User Guide and be familiar with all instrument safety information. Before using the MGS1 System, read the Safety and EMC Compliance section in the MGS1 System Hardware Guide and be familiar with all instrument safety information. 1 Product Description Instrument Safety Materials Preparing the THAP Matrix Preparing the Primer Extension Mixture Applying Matrix and Primer Extension Mixture to the Voyager Sample Plate Testing the MGS1 System Checking and Optimizing Resolution and Sensitivity Troubleshooting Storing the Kit Accessories, Spare Parts, and Ordering Information Technical Support

2 4 Preparing the THAP Matrix WARNING: CHEMICAL HAZARD. Matrix Diluent (with acetonitrile) is a flammable liquid and vapor. It may cause eye, skin, and respiratory tract irritation, central nervous system depression, and heart, liver, and kidney damage. Please read the MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. WARNING: CHEMICAL HAZARD. 2,4,6 trihydroxyacetophenone (THAP) may cause eye, skin, and respiratory tract irritation. Please read the MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Preparing CAUTION: To prevent contamination, wear gloves when performing the following steps, do not touch the internal surfaces of the vials or caps, and do not allow the underside of the caps to touch any surfaces. To prepare the THAP matrix: 1. Transfer the contents of one of the vials of matrix diluent to the vial of THAP matrix powder. 2. Cap and vortex the mixture for 5 to 10 seconds. 3. Allow any undissolved matrix to settle. Matrix Stability Protect THAP matrix powder from high temperatures and light. Discard THAP matrix solution if it turns dark brown or fails to crystallize. For more information, see Section 10, Storing the Kit. 5 Preparing the Primer Extension Mixture WARNING: CHEMICAL HAZARD. Matrix Diluent (with acetonitrile) is a flammable liquid and vapor. It may cause eye, skin, and respiratory tract irritation, central nervous system depression, and heart, liver, and kidney damage. Please read the MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. WARNING: CHEMICAL HAZARD. The toxicological properties of Primer Extension Mixture have not been thoroughly investigated. Use appropriate precautions. Read the MSDS. Preparing CAUTION: To prevent contamination, wear gloves when performing the following steps, do not touch the internal surfaces of the vials or caps, and do not allow the underside of the caps to touch any surfaces. To prepare the primer extension mixture: 1. Immediately before use, transfer 25 µl matrix diluent to the vial of primer extension mixture. 2. Cap and vortex the mixture for 5 to 10 seconds. Primer Extension Mixture Stability The reconstituted primer extension mixture is stable for approximately 3 days at room temperature. For more information, see Section 10, Storing the Kit. 6 Applying Matrix and Primer Extension Mixture to the Voyager Sample Plate 6.1 Overview The following sections describe how to manually spot the THAP matrix and primer extension mixture on a Voyager sample plate. NOTE: If you use this kit to test the MGS1 System, do not use a 96 2-position, flat, hydrophobic plastic surface plate. Use only a sample plate supported by the MGS1 software, such as: 96-position, barcoded 100-position 384-position 384-position, barcoded 400-position 6.2 Cleaning the Sample Plate WARNING: CHEMICAL HAZARD. Acetonitrile is a flammable liquid and vapor. It may cause eye, skin, and respiratory tract irritation, central nervous system depression, and heart, liver, and kidney damage. Please read the MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. CAUTION: Do not scrub or sonicate the Voyager sample plate. Scrubbing or sonicating can damage the coating on the plate. Before spotting, clean the sample plate: 1. Rinse the sample plate with deionized or Milli-Q (not distilled) water to remove previous samples and contaminating salts. 2. If the sample plate contains analytes or matrixes that are not water soluble, rinse the plate with acetonitrile, then rinse with deionized or Milli-Q water. 3. Allow to air-dry. 6.3 Spotting the Sample Plate CAUTION: To prevent contamination, wear gloves when performing the following steps, do not touch the internal surfaces of the vials or caps, and do not allow the underside of the caps to touch any surfaces. To spot the sample plate: 1. In a fresh 200-µL MicroAmp tube, mix 5 µl THAP matrix solution with 5 µl reconstituted primer extension mixture. 2. Spot two or three positions on the Voyager sample plate with the 50:50 THAP matrix:reconstituted primer extension mixture. The volume you spot depends on the sample plate you use: Sample Plate 3. Dry the plate in either of the following ways: Spot Volume (µl) 384- and 400-position sample plates 0.4 to and 100-position sample plates 1.0 Air-dry at room temperature for a few minutes. Place in a stream of gently flowing air, for example, in the airstream of a fume or laminar-flow hood. IMPORTANT: Do not use the air supply at a lab bench to dry the plate unless the air supply is free of oils and additives. 2

3 6.4 Sample Stability Dried samples on a sample plate are stable for approximately one day. Protect the plate from light if you do not analyze the samples within an hour of spotting. 7 Testing the MGS1 System 7.1 Overview The following sections describe how to use the DNA Standards Kit to check that the MGS1 System (excluding the SymBiot XT Workstation) is operating properly and has instrument settings optimized to successfully identify alleles. It includes: Before you begin Creating a primer Creating a sample set Acquiring and processing data Viewing results 7.2 Before You Begin Before you begin, complete the steps in: Section 4, Preparing the THAP Matrix Section 5, Preparing the Primer Extension Mixture Section 6, Applying Matrix and Primer Extension Mixture to the Voyager Sample Plate 7.3 Creating a Primer To create a primer: 1. Open the MGS1 software by double-clicking. 2. From the View menu, select Primers and Pools. 3. Click to display the Primer Browser. 4. If the R DNA Standard primer already exists in the Primer Browser, go to Section 7.4. If not, continue with step In the Primer section at the top of the screen, click New. 6. For the Description, type DNA Standards Kit Primer. 7. In the Primer Sequence (5 to 3 ) text box, type: CTTTGTTCTGGGTTTC A calculated mass of appears in the Mass text box. 8. In the Expected Base Added to Primer text box, type AT. 9. Click Save. In the Primer Browser, type R DNA Standard for the Name, then click Save. 7.4 Creating a Sample Set To create a sample set: 1. In the MGS1 software, select Sample Set from the View menu. 2. At the top of the Sample Set screen, click New, then select the following general information: 3. In the Select MALDI Plates section, click to display the Plate Browser. 4. In the Plate Browser, click to display the Plate Editor. 5. In the Plate Name text box, type DNA Standard Plate, click OK, then click OK again. 6. In the Set All Plates tab, click Add All to Sample List to add the DNA Standard Plate you created in step 5 to the Sample Set List. 7. In the Sample Set List, type or select the following information for the positions that contain the primer extension mixture. 8. Click-drag to select all plate positions that you did not spot, then click above the Sample Set List. 9. In the Analysis Setup section, click Acquisition Method. 10. In the Acquisition Methods dialog box, select DNA Standard Kit from the Acquisition Method Name list. The DNA Standard Kit Acquisition Method specifies: Data File Directory Voyager Computer Name\MGS1\Data BIC File Name Voyager Computer Name\MGS1\ BIC_SET_Files\DNAStdKit.BIC or DNAStdKitPRO.BIC NOTE: Open the DNAStdKit.BIC or DNAStdKitPRO.BIC file in the Voyager Instrument Control Panel and verify the Control Mode is set to Automatic. If it is not, change the Control Mode to Automatic, then save the file. 11. Click OK. Parameter Setting Sample Origin MALDI Plate MALDI Plate Type (Select the MALDI Plate Type that matches the plate you spotted in Section 6, Applying Matrix and Primer Extension Mixture to the Voyager Sample Plate.) Parameter Sample Name Single Primer Primer/Pool Name Selection DNA Standard Select R DNA Standard 12. In the Analysis Setup section, click Processing Method. 13. In the Processing Methods dialog box, select DNA Standard Kit from the Processing Method Name list. The DNA Standard Kit Processing Method specifies the settings shown in Table 1 on page Click OK. 3

4 Table 1 DNA Standard Processing Method Settings Parameter 15. Click Save at the top left of the Sample Set screen, type DNA Standard Sample Set in the Sample Set Selection text box, then click OK. 7.5 Acquiring and Processing Data Selecting an Acquisition Mode and Sample Plate To select an acquisition mode and sample plate: 1. If the Control Panels are not displayed in the MGS1 software, select Control Panels from the View menu. 2. In the Data Acquisition Control Panel, select Acquire and Process Data for the Mode. 3. Click to display the Select Plate dialog box. Setting Processing Settings Smoothing Methods Noise Filter Correlation Factor 0.7 Baseline Correction Advanced Data Explorer SET File Voyager Computer Name\ MGS1\BIC_SET_Files\ DNAStdKit.SET Calibration Settings External Calibration File (Not applicable, leave blank) Peaks for internal calibration Primers Only Relative Peak Intensity (%) 20 Mass Tolerance (Da) 15 Max Outlier Error (m/z) 2 Base Calling Settings Allele Tolerance (Da) 1.5 Heterozygote Threshold (%) Select DNA Standard Plate, then click OK to add it to the Data Acquisition Control Panel. 4. In the Load/Eject dialog box, click Eject to eject the plate holder. CAUTION: Verify that the sample plate is completely dry before loading. Loading a wet plate can cause vacuum errors and instrument damage. 5. Slide the sample plate into the holder from the right side with the slanted underside of the plate facing to the left and toward the back of the instrument, then snap the plate into place. 6. From the Sample Plate menu, select Load to retract the sample plate and insert it into the main source chamber. 7. Select a Plate ID with a.plt file that corresponds to the Voyager sample plate you used to spot the THAP matrix and primer extension mixture. 8. Click Load to load the sample plate. Wait for the Load/Eject Status dialog box to close. 9. Align the sample plate by selecting Align from the Sample menu. For more information on aligning the sample plate, see the MGS1 System Hardware Guide. Starting Acquisition After the Load/Eject Status dialog box closes in the Voyager software, return to the MGS1 computer and click OK. CAUTION: Do not click OK until the Load/Eject Status dialog box closes. If you click OK before the mass spectrometer completes the Load/Eject cycle, invalid results can occur. 7.6 Viewing Results To view results after acquisition is complete: 1. In the MGS1 software, open the DNA Standard Sample Set by selecting Sample Set from the View menu, clicking to display the Sample Set Browser, selecting DNA Standard Sample Set, then clicking OK. 2. Click the Result Viewer tab. 3. Select DNA Standard Plate from the Plate list, then select R DNA Standard from the Primer list. The results for the DNA Standard Plate and the R DNA Standard primer are displayed in the Result Viewer tab (Figure 2). Loading the Sample Plate in the Voyager Workstation WARNING: LASER HAZARD. Lasers emit ultraviolet radiation. Lasers can burn the retina and leave permanent blind spots. Never look directly into the laser beam. Remove jewelry and other items that can reflect the beam into your eyes. Do not remove the instrument front or side panels. Wear proper eye protection and post a laser warning sign at the entrance to the laboratory if the front or side panels are removed for service. Green = A Red = T To load the sample plate in the Voyager Workstation: 1. Power up the Voyager Workstation and start the Instrument Control Panel. 2. In the Data Acquisition Control Panel, click. A message prompts you to load the sample plate in the Voyager mass spectrometer. 3. In the Voyager Instrument Control Panel, select Eject from the Sample Plate menu. Figure 2 Results of DNA Standard Plate and R DNA Standard Primer 4

5 4. Check that the positions on the plate that contain the THAP matrix and primer extension mixture are green and red, indicating an A/T heterozygote for the extended primers. 5. Click a position on the plate that contains the THAP matrix and primer extension mixture to display the Results List (Figure 3). Results of Primer Extension Mixture 8.3 Optimizing the Voyager Workstation (Laser Intensity) WARNING: LASER HAZARD. Lasers emit ultraviolet radiation. Lasers can burn the retina and leave permanent blind spots. Never look directly into the laser beam. Remove jewelry and other items that can reflect the beam into your eyes. Do not remove the instrument front or side panels. Wear proper eye protection and post a laser warning sign at the entrance to the laboratory if the front or side panels are removed for service. Before checking resolution and sensitivity, optimize the Voyager Workstation settings from the Instrument Control Panel: 1. Power up the Voyager Workstation and start the Instrument Control Panel. CAUTION: Verify that the sample plate is completely dry before loading. Loading a wet plate can cause vacuum errors and instrument damage. Figure 3 Results List for Primer Extension Mixture If your results do not indicate an A/T heterozygote, see Section 9, Troubleshooting. 8 Checking and Optimizing Resolution and Sensitivity 8.1 Overview The following sections describe how to check and optimize resolution on the Voyager Workstation to optimize it for DNA analysis. It includes: Before you begin Optimizing the Voyager Workstation (laser intensity) Checking resolution and sensitivity Resolution requirements 8.2 Before You Begin Before you begin, complete the steps in: Red = T Green = A Section 4, Preparing the THAP Matrix Section 5, Preparing the Primer Extension Mixture Section 6, Applying Matrix and Primer Extension Mixture to the Voyager Sample Plate 2. Load the sample plate in the Voyager Workstation. For the Plate Type, select or create a Plate ID with a.plt file that corresponds to the Voyager sample plate you used to spot the THAP matrix and primer extension mixture in Section 6, Applying Matrix and Primer Extension Mixture to the Voyager Sample Plate. 3. Click in the toolbar of the Instrument Control Panel to turn on the high-voltage power supplies. Allow the high-voltage power supplies to warm up for 30 minutes for maximum mass accuracy. 4. From the Voyager Workstation Instrument Control Panel, open the DNAStdKit.BIC or DNAStdKitPRO.BIC file, located on the Voyager computer in the D:\MGS1\BIC_SET_Files subdirectory. (Open the.bic file appropriate for your instrument, Voyager-DE or Voyager-DE PRO Workstation.) Verify the Control Mode is set to Manual. If you do not have an MGS1 System, create the DNAStdKit.BIC or DNAStdKitPRO.BIC file using the settings shown in Table 2. Table 2 Instrument Settings for DNAStandardsKit.BIC File Parameter Setting for Voyager-DE Workstation Instrument Mode Linear Linear Extraction Type Delayed Delayed Polarity Type Positive Positive Linear Digitizer (Acqiris) Bin Size 2 nsec 2 nsec Setting for Voyager-DE PRO Workstation Vertical Scale 1000 mv 500 mv Vertical Offset Input Bandwidth Full Full Control Mode Manual Manual Accelerating Voltage 20,000 V 20,000 V Grid Voltage% 94.5% 94.0% Guide Wire Voltage% 0.05% 0.15% Extraction Delay Time 300 nsec 350 nsec Laser Shots/Spectrum Acquisition Mass Range 3,000 to 7,000 Da 3,000 to 7,000 Da Low Mass Gate 3,000 Da 3,000 Da Calibration Matrix THAP THAP Calibration File Default Default 5

6 5. In the Manual Laser Intensity section of the Instrument Control Panel, set the laser intensity to 2, Use the Manual Sample Positioning section of the Instrument Control Panel to select a position on the sample plate containing the THAP matrix and primer extension mixture. 7. Start acquisition by clicking on the toolbar. 8. During acquisition, view the spectrum in the Instrument Control Panel. Verify that it looks similar to Figure 4. Unextended primer peak Depurination peaks Primer extension peaks Figure 4 Spectrum of Primer Extension Mixture 9. Increase the laser intensity in 50-step increments and observe the signal intensity of the 4,860 Da peak. You typically see major changes in signal intensity between laser intensity settings of 2,000 and 2,100. At a laser intensity setting between 2,100 and 3,000, you typically see a plateau in which changes in signal intensity do not occur when you increase the laser intensity. 10. Continue increasing the laser intensity in 50-step increments until the signal intensity starts to decrease and you see peak broadening and fragmentation. The optimum laser intensity setting is midway between the intensity that yields the signal intensity plateau and the intensity that causes peak broadening and fragmentation (Figure 5). 8.4 Checking Resolution and Sensitivity To check resolution and sensitivity: 1. Optimize the Voyager Workstation settings as described in Section 8.3, Optimizing the Voyager Workstation (Laser Intensity). 2. In the Data Storage section of the Instrument Control Panel, specify D:\Voyager\Data as the directory for storing the data. 3. Use the Manual Sample Positioning section of the Instrument Control Panel to select a position on the sample plate containing the THAP matrix and primer extension mixture. 4. With the DNAStandardsKit.BIC or DNAStandardsKitPRO.BIC file open, start acquisition by clicking in the toolbar. Acquire five spectra from different regions of the same spot, saving each spectrum to a data file. 5. From the Tools menu in the Voyager Workstation Instrument Control Panel, use the Resolution Calculator and the Signal-to-Noise Calculator to calculate the resolution and signal-to-noise ratio of the unextended primer peak (4,860 Da) in all five data files, then average the results. NOTE: When calculating signal-to-noise ratios, specify a Baseline Region that is flat (non-rising) and does not include peaks. 6. Check that the resolution is adequate to support your application. See Table 3. The signal-to-noise ratio is typically 50:1. 7. If resolution is not adequate, do any of the following: Adjust laser intensity as described in Section 8.3, Optimizing the Voyager Workstation (Laser Intensity). Adjust Delay Time in 50-nsec increments. Adjust Guide Wire Voltage% in 0.05% increments. NOTE: Adjusting laser intensity and Delay Time to optimize resolution typically also improves the signal-to-noise ratio. 8.5 Resolution Requirements The instrument resolution required to resolve allelic pairs is a function of the mass of the oligonucleotide containing the two alleles. The mass of the oligonucleotide is proportional to its length (number of bases). See Table 3. Major changes in signal intensity occur with minor laser setting adjustments Plateau, no changes in signal intensity occur with laser setting adjustments Peak broadening and fragmentation occur at higher laser settings Table 3 Maximum Primer Length (Number of Bases) Supporting Resolution of Allelic Pairs Resolution Allelic Pair Mass Signal Intensity Optimum setting midway between plateau setting and setting that causes peak broadening and fragmentation ~2000 ~2500 Laser Intensity Figure 5 Determining Optimum Laser Intensity A/C A/G A/T C/G C/T G/T With the laser intensity at its optimum setting, save the DNAStandardsKit.BIC file. The laser intensity setting is saved with the.bic file. For more information on optimizing settings, see the Voyager Biospectrometry Workstation User Guide, Section 5.1.4, Modifying an Instrument Settings File (.BIC). Table 3 serves as a guide for determining if the resolution of your mass spectrometer is adequate to support a given application. For example, at a measured resolution of 500 (m/ m), the longest primer containing a C/T heterozygote that can be resolved is 24 bases long. If the allelic pair to resolve is unknown, assume you need a resolution that can resolve the allelic pair with the lowest mass difference (A/T). 6

7 NOTE: Primers over 30 bases in length are not recommended, even if resolution requirements would support their use. Instrument response decreases with longer primers. 9 Troubleshooting Use Table 4 to troubleshoot problems you may encounter when testing the MGS1 System. Table 4 Troubleshooting Testing of the MGS1 System Symptom Possible Cause Action No result in the Result Viewer tab of the MGS1 software. The results in the Result Viewer tab of the MGS1 software indicate a single allele (an A or T homozygote). The mass scale of the Voyager Workstation is not calibrated. The resolution or signal-to-noise ratio of the Voyager Workstation is not optimized. The Heterozygote Threshold (%) setting in the Processing Method is too high. 1. Open the data file in the Data Explorer software. 2. Create an external calibration file (.CAL). 3. In the MGS1 software open the DNA Standard Processing Method and select this external calibration file (.CAL). 4. Reprocess the data. Adjust laser intensity and Delay Time to improve resolution and sensitivity. See Section 8, Checking and Optimizing Resolution and Sensitivity. Decrease the Heterozygote Threshold (%) setting. 11 Accessories, Spare Parts, and Ordering Information To order accessories and spare parts, contact Applied Biosystems Customer Service. Refer to the back page of this document for the phone number and web address. Description 12 Technical Support Quantity Part Number Sequazyme DNA Standards Kit 1 kit Sequazyme Pinpoint SNP Assay Kit 1 kit, provided in four separate boxes Voyager sample plate, hydrophobic plastic surface, flat, 384-position Voyager sample plate, hydrophobic plastic surface, flat, 384-position, barcoded 1 plate V plate For technical assistance, call Applied Biosystems Technical Support. From North American, dial If you are outside North America, refer to the back page of this document for the phone number and web address. 10 Storing the Kit The DNA Standards Kit is most stable at 20 C. Kit components are less stable at higher temperatures (see table below). Avoid prolonged exposure to light. Kit Component Storage Temperature Stability Unopened kit 20 C 1 year THAP Matrix (powder) 20 C 1 year Room temperature 1 month THAP Matrix 20 C 1 week (reconstituted) Room temperature 1 day Matrix Diluent 20 C 1 year Primer Extension Mixture (dried) <4 C 1 year Primer Extension Mixture (reconstituted) 20 C 1 month 4 C 1 week Room temperature 3 days 7

8 Copyright 2001, Applied Biosystems All rights reserved For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document. Applied Biosystems, SymBiot, and MicroAmp are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. Headquarters 850 Lincoln Centre Drive Foster City, CA USA Phone: Toll Free (In North America): Fax: Worldwide Sales and Support Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches into 150 countries on six continents. For sales office locations and technical support, please call our local office or refer to our web site at AB (Design), Applera, Biospectrometry, MassGenotyping Solution 1, Sequazyme, Voyager, and Voyager-DE are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. Milli-Q is a registered trademark of Millipore Corporation. All other trademarks are the sole property of their respective owners. Applera Corporation is committed to providing the world s leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses. Printed in the USA, 04/2001 Part Number Rev. A