tay sachs application note€¦ · tay sachs disease (tsd) is a genetic neurodegenerative lysosomal...

TAY SACHS Application Note

of 26

02-01/2013 140959.TS

TAY SACHS Application Note (For use with NanoChip® 400 Instrument)

Savyon Diagnostics Ltd. 3 Habosem St. Ashdod 77610 ISRAEL Tel.: +972.8.8562920 Fax: +972.8.8523176 E-mail: [email protected]

mailto:[email protected]


of 26

02-01/2013 140959.TS

European Authorized Representative: Obelis s.a. Boulevard Général Wahis 53 1030 Brussels, BELGIUM Tel: +(32) 2. 732.59.54 Fax: +(32) 2.732.60.03 E-Mail : [email protected]

Table of Contents

Introduction ......................................................................................... 3 Intended Use ................................................................................................ 3 Background .................................................................................................. 3 Kit Contents .................................................................................................. 3 Storage ....................................................................................................... 3 Using NanoChip Cartridges ............................................................................... 4

NanoChip Cartridge Handling ....................................................................... 4

Materials and Equipment ......................................................................... 4 Materials Available from.................................................................................... 4 Additional Materials Available from Savyon ............................................................. 5 Other Required Materials (not available from Savyon ) ................................................ 5 Required Equipment ........................................................................................ 5

Technical Assistance ............................................................................... 6

Precautions ......................................................................................... 7

Performing Sample Amplification ............................................................... 8 Extraction .................................................................................................... 8 Amplification ................................................................................................. 8 Preparing the Sample Plate .............................................................................. 10

Operating the NanoChip 400 System ........................................................... 12 Preparing Solutions for Use in the NanoChip 400 Instrument ....................................... 12 Preparing the NanoChip Cartridge and Instrument .................................................... 13 Creating a Protocol ........................................................................................ 14 Running the Assay ......................................................................................... 16 Analyzing the Data ......................................................................................... 18

Appendix A: TAY SACHS Assay Format ........................................................ 19

Appendix B: TAY SACHS Data Analysis Spreadsheet and Data Calculations .............. 22

Appendix C: Legal Notices ...................................................................... 25 REFERENCES ................................................................................................ 26

mailto:[email protected]


of 26

02-01/2013 140959.TS

Introduction

Intended Use

The TAY SACHS Kit is used to detect and identify a panel of six genetic mutations in the human HEXA gene. The amplified DNA is tested for the following mutations: 1278 InsTATC, IVS12 +1G>C, G269S, R170Q, DF304/305 and IVS5-2 A>G. For professional use only

Background

TAY SACHS Disease (TSD) is a genetic neurodegenerative lysosomal storage disorder. TSD is fatal in its most common variant known as Infantile Tay-Sachs disease. TSD is inherited in an autosomal recessive pattern. The disease occurs when fatty acid derivative called ganglioside accumulate in the nerve cells of the brain due to deficiency in the activity of the Hexozaminidase A (Hex A) enzyme (2). TSD is caused by mutations on the HEXA gene on chromosome 15. The carrier frequency of TSD is 1:29 in Ashkenazi Jews 1:110 in Moroccan Jews and 1:280 in the general Jewish Israeli population (1).

Mutations Gene

1278 InsTATC HEXA

IVS12 +1G>C HEXA

G269S HEXA

R170Q HEXA

DF 304/305 HEXA

IVS5-2 A>G HEXA

Table 1: Mutations detected by TAY SACHS Kit.

Kit Contents

The TAY SACHS Kit contains enough amplification buffer and primer mix for 96 or 192 samples and enough detection reagents for three detections run. One to 96 samples can be analyzed in a single detection run. Refer to product package insert for performance characteristics and additional storage information.

Storage

REF 800572 800575

≤-20°C ≤-20°C


of 26

02-01/2013 140959.TS

Using NanoChip Cartridges

The TAY SACHS Kit is designed to analyze 96 or 192 samples on a NanoChip 400 Cartridge. A maximum

of six TAY SACHS protocols may be run on a NanoChip 400 Cartridge.

NanoChip Cartridge Handling Handle the cartridge by the outer black housing only; do not touch the clear plastic or electrical contact

area. Exposure to static electricity may damage the cartridge and may affect results. Ensure that the

flowcell window (clear plastic on the underside of the cartridge) is clear of any debris. If debris is present,

always use a new (not previously opened) Bausch & Lomb Pre-Moistened Tissue to clean the window. DO

NOT use excessive force when wiping the flowcell window. ONLY clean the flowcell window if debris is

present.

Materials and Equipment

Materials Available from

REF Description Contents

FA800572 TAY SACHS Kit 96 Samples

TAY SACHS Amplification Reagents 1 x vial (624 µL) Primer Mix 2 x vial (850 µL) LS Amplification

Buffer TAY SACHS Reagent Packs

2 x Cap/Rep Reagent Pack

1 x bottle CAPdown Sample Buffer B

FB800572 TAY SACHS Kit 192 Samples

TAY SACHS Amplification Reagents 2 x vial (624 µL) Primer Mix 4 x vial (850 µL) LS Amplification

Buffer TAY SACHS Reagent Packs

2 x Cap/Rep Reagent Packs

1 x bottle CAPdown Sample Buffer B

800575 TAY SACHS Cap/Rep Reagent Packs

4 x Cap/Rep Reagent Packs


of 26

02-01/2013 140959.TS

Additional Materials Available from Savyon

REF Description Contents

800160 NanoChip 400 Cartridge 1 cartridge

800161 NanoChip 400 Fluidics Cartridges 4 x fluidics cartridges

800154 NC400 Low Salt Buffer 4 x bottles (25 mL each)

800155 NC400 High Salt Buffer 4 x bottles (25 mL each)

800156 NC400 Target Prep Buffer 4 x bottles (25 mL each)

800061 NanoChip Microplate Seals 100 x 96-well plate seals

Other Required Materials (not available from Savyon )

Extraction Reagents:

Reagents to extract genomic DNA from blood at a yield > 50 ng/μL

Amplification Reagents: FastStart Taq DNA polymerase (Roche) Cat# 04 738 420 001

Reagents to run NanoChip® 400 system: L-histidine (Sigma H-8000) Triton® X-100 (Sigma X-100) Water, deionized

Sample Plates

96-well ABI PCR plates (ABI N801-0560)

96-well Thermo-Fast PCR plates (AB-1100)

MicroAmp™ Compression Pads (ABI 4312639)

2µm filters (Nalgene 5660020)

Required Equipment

NanoChip 400 System

Thermal Cycler1

1 The following models are recommended:

GeneAmp® Thermal Cycler 2700, 2720, or 9700 MJ Research Peltier Thermal Cycler PTC200


of 26

02-01/2013 140959.TS

Technical Assistance

Specialists from the Technical Assistance Center can help troubleshoot and resolve problems. Contact the Center via one of the following methods:

E-mail: [email protected]

Phone: +972.8.8562920

Fax: +972.8.8523176

Address: Savyon Diagnostics Ltd.

3 Habosem St. Ashdod 77610 ISRAEL


of 26

02-01/2013 140959.TS

Precautions

Amplification technologies can amplify target nucleic acid sequences over a billion-fold and

provide a means of detecting very low concentrations of target. Care must be taken to

avoid contamination of samples with target molecules from other samples, or amplicons

from previous amplifications. Follow these recommendations to help control

contamination.

1. If possible, isolate pre-amplification steps from post-amplification steps. For example, use separate

rooms for pre- and post-amplification. Each room should contain equipment, such as pipettes,

dedicated to the specific process. Gloves and lab coats should be dedicated to each room as well.

If dedicated rooms are not available, the laboratory should be set up to allow a unidirectional flow.

Prepare samples in a laminar flow hood using dedicated equipment to minimize contamination. Set

up the post-amplification area in a low-traffic area with dedicated equipment.

2. Use disposable containers, disposable barrier pipette tips, disposable bench pads, and disposable

gloves. Avoid washable lab wear.

3. Use a diluted bleach solution (0.2% sodium hypochlorite) to treat waste from the post-amplification

and detection areas, as the waste contains amplicon. Use the bleach solution to wipe down

equipment and bench areas, and to treat drains used to dispose of liquid waste.

4. Monitor contamination with regular swabbing. Use a wet cotton swab to wipe areas of the bench or

equipment, and rinse the swab with 500 µL of water. Test a few microliters of the rinse solution in

the amplification assay to detect possible contamination. If contamination is detected, follow internal

de-contamination procedures.

5. Use negative controls to monitor for possible contamination during reaction setup. If reagent

contamination is detected, dispose of the suspect reagents.

References for Contamination Control

Kwok, S. and Higuchi, R. (1989). Avoiding false positives with PCR. Nature (London) 339, 237.

Victor, T. et al. (1993). Laboratory experience and guidelines for avoiding false positive polymerase chain reaction results. Eur. J. Clin. Chem. Clin. Biochem. 31, 531.

Yap, E.P.H. et al. (1994). False-positives and contamination in PCR. In: PCR Technology: Current Innovations. Griffin, H.G. and Griffin, A.M., eds., CRC Press, Boca Raton, FL.


of 26

02-01/2013 140959.TS

Performing Sample Amplification

To optimize workflow, you can begin other activities during sample amplification. For example, you can prepare the system and thaw reagents. During cartridge initialization, you can write the protocol and prepare the sample plate.

Extraction

Process a blood sample using an extraction method that yields ≥ 50 ng/µL of genomic DNA.

Amplification

Perform in an amplicon-free area. 1. Remove the LS Amplification Buffer and the Tay Sachs Primer Mix from the ≤ -20°C freezer.

Thaw at room temperature and vortex.

Note: The LS Amplification Buffer and the Tay Sachs Primer Mix may be frozen two additional times, or stored at 2-8º C for one week.

2. Prepare the PCR Master Mix using the following guidelines per sample

(see Table 2). To ensure an adequate volume of Master Mix, take the number of reactions and add 2. Multiply the sum by the volume of each component shown in Table 1.

Note: Remove the FastStart Taq DNA Polymerase from

the freezer immediately prior to use, and return to the freezer promptly after use.

Table 2: PCR

1 Master Mix Guidelines

Component Volume (µL)

LS Amplification Buffer 12.5

Tay Sachs Primer Mix 6.5

FastStart Taq DNA Polymerase 1.0

Total Master Mix Volume per Reaction 20

3. Add 20 µL of the PCR Master Mix to each reaction well in the PCR plate. 4. Add 5 µL of template DNA to each reaction well.

1 Refer to Appendix C: Legal Notices, for PCR information.


of 26

02-01/2013 140959.TS

Notes: Do not scale up an amplification reaction; always use 25 µL reaction volumes.

Template DNA must be at least 50 ng/µL. 5. Seal the PCR plate and place into a thermal cycler.

Note: For onboard dilution, cover the 96-well ABI PCR

plate with a Nanochip Microplate seal (PN: 800061) and place the plate into the thermal cycler

1.

Place the ABI MicroAmp Compression Pad over the sealed PCR 96-well plate and close the lid of the thermal cycler. Alternatively, the 96-well ABI PCR

plate may be

sealed with standard PCR caps. The caps must be removed and replaced with a Nanochip Microplate seal (PN: 800061) prior to use on the NanoChip 400.

6. Program the thermal cycler using the parameters described in Table 3.

Table 3: Thermal Cycler Parameters

Temperature (°C) Time Number of Cycles

95 2 minutes 1

95 30 seconds

40 65 1 minute

72 7 minutes 1

4 Hold

7. Once cycling is complete, remove the PCR plate from the thermal cycler.

The prepared plate may be stored at 2-8°C for up to one week, or at ≤ -20°C for up to six months.

1 The following models are recommended:

GeneAmp® Thermal Cycler 2700, 2720, or 9700 MJ Research Peltier Thermal Cycler PTC200


of 26

02-01/2013 140959.TS

Preparing the Sample Plate

Notes: To optimize workflow, begin system preparation, reagent thawing, and creating the

protocol during sample amplification. If not using onboard dilution, prepare the sample plate during cartridge initialization.

The sample dilution option must be set in the Tay Sachs template in the Protocol

Editor such that the PERFORM ONBOARD DILUTION setting is checked for automated onboard dilution and unchecked for manual dilution. The template default has the PERFORM ONBOARD DILUTION setting checked for automated onboard dilution.


of 26

02-01/2013 140959.TS

Option 1 Manual Sample Dilution

1. Remove CAPdown Sample Buffer B from the freezer. Upon thawing, vortex the solution thoroughly

until all precipitates are dissolved.

Note: Once thawed, CAPdown Sample Buffer B can be stored at room temperature or at 2-8° C for up to two weeks. Do not refreeze.

2. For each individual amplification reaction, pipette 62 µL of CAPdown Sample Buffer B into one well of

a 96-well Nunc plate.

3. Add 8 µL of each amplification reaction into a well containing CAPdown Sample Buffer B. Carefully

pipette up and down to mix. 4. Cover plate with a Microplate Seal.

Option 2 Onboard Sample Dilution

1. Remove the ABI MicroAmp™ Compression Pad from the ABI PCR plate covered with Microplate

Seal, attach the plate to the PCR Plate Base and insert into plate position 2 of the NanoChip 400. Or

1. Remove the caps of the ABI PCR plate and replace with a Microplate Seal, attach the plate to the PCR Plate Base and insert into plate position 2 of the NanoChip 400.

Or

1. Pipette a minimum of 20 μL of amplified sample into wells of a NUNC 96 well plate 2. Cover with a Microplate Seal and insert plate into plate position 2 of the NanoChip 400.

Note: The Onboard Dilution Option can only be used with the ABI 96 well plate (ABI

N801-0560) attached to the PCR Base Plate or with the NUNC 96 well plate (NUNC 249944). Use of other plate types may cause damage to the instrument.


of 26

02-01/2013 140959.TS

Operating the NanoChip 400 System

Refer to the NanoChip 400 User’s Guide (REF 140530) for detailed instructions on the basic operation of the system, including system maintenance and cartridge handling.

Preparing Solutions for Use in the NanoChip 400 Instrument

The following table describes the required solutions, and their assigned locations within the instrument.

Table 4: Location of Bottles in the NanoChip 400 Instrument

Solution Bottle Location Minimum Volume*

Water 1 L H2O position 400 mL

Wash Solution 1 L BUF position 400 mL

High Salt Buffer 30 mL Position 1 25 mL

Low Salt Buffer 30 mL Position 2 25 mL

Target Prep Buffer 30 mL Position 3 25 mL

**CAPdown Sample Buffer B

30 mL Position 4 25 mL

* The minimum volume of liquid that should be in the listed bottle before starting the

assay run

**CAPdown Sample Buffer B is only required if performing onboard dilution.

Preparing the Wash Solution Preparing the Wash Solution for NC400 Instrument.

1. 50 mM histidine solution

In a bottle/beaker, add 7.76 g of L-histidine to a final volume of 1 L of dH2O for 50 mM histidine. Mix until histidine is dissolved. Filter

the solution using a 0.2 m filter.

Note: This solution is stable for up to two week at 2–8oC.

2. 20% Triton X-100 solution

a. Add 4 mL or 4.24 g of Triton X-100 to approximately 16 mL of dH2O for a final volume of 20 mL.

b. Mix solution thoroughly (approximately 10 minutes).

Note: This solution is stable for up to three months at 2-8oC.

3. Combine component solutions daily to make fresh wash solution

(50 mM histidine, 0.1% Triton X-100).


of 26

02-01/2013 140959.TS

a. Add 400 mL of the 50 mM histidine solution to a 1 L buffer

bottle. b. Add 2 mL of the 20% Triton X-100 solution and mix

thoroughly. Note: Make wash solution fresh daily.

Preparing the NanoChip Cartridge and Instrument

1. Remove the following reagent pack from the freezer and place at room temperature

to thaw.

TAY SACHS Cap/Rep Reagent Pack Notes: The reagent pack must be used within 8 hours of thawing.

Because the item listed above is single use only, discard after use.

2. Remove a NanoChip Cartridge from 2-8°C storage. Keep at room temperature for at least 15 minutes before using. Note: Bringing the cartridge to room temperature before insertion into the

instrument avoids the formation of condensation in the cartridge window, which could cause the cartridge to fail initialization.

3. Initialize and prime the NanoChip 400 Instrument following the guidelines listed in

the NanoChip 400 User’s Guide. 4. From the Dock Bar, select the instrument icon to start the NanoChip 400 Instrument

Manager.

5. Ensure that the flowcell window (clear plastic on the underside of the cartridge) is clear of any debris. If debris is present, use a new (not previously opened) Bausch & Lomb Pre-Moistened Tissue to clean the window.

Note: Do NOT use excessive force when wiping the flowcell window. Clean the

flowcell ONLY when debris is present.

6. Scan the barcode of the NanoChip Cartridge using the attached barcode scanner.

Note: The barcode will not display in the Instrument Manager until step 8 has been completed.

7. Insert the cartridge into the instrument, ensuring that it is properly seated.

8. Close the cartridge door by pressing the button located below the cartridge slot on the instrument.


of 26

02-01/2013 140959.TS

9. When the Cartridge Initialization window appears, select Initialize Cartridge with Hydration.

10. Cartridge initialization will take approximately 15 minutes. When initialization is

completed, the LCD will display “Instrument Ready”.

11. Write the protocol as described in the following section.

Note: The protocol can be written while the cartridge is initializing.

Creating a Protocol

Using the Protocol Editor, create the following protocol to address and report 1-96 samples.

Create a new protocol for each sample run. For detailed instructions on using Protocol

Editor, see the NanoChip 400 User’s Guide.

1. From the Dock Bar select Protocol Editor.

2. Select Create A New Protocol; select OK.

3. Select the TAY SACHS icon from the available templates (see Figure 1).

Note: The TAY SACHS template automatically determines prior pad utilization, and maps capture and sample addressing beginning with the first unused sample position.

4. The Plate Specification Window appears; choose the correct plate type intended for the assay from the options in the pull-down menu. Select OK.

Note: Selecting a sample plate type other than what is placed on the NanoChip 400 Instrument deck at the start of a run can cause damage to the system and fail the run. Use caution to select the appropriate plate type.

5. The Set Cartridge window appears; choose Select The Cartridge. From the pull-down menu, select the serial number of the cartridge that will be used in the run (or type the serial number into the window). Select OK.

Note: If the cartridge selected is still initializing, a cartridge presently in use window will appear. Select Yes to indicate that you still want to use this cartridge for the protocol you are creating.

Warning: Select No if the cartridge selected is in use with a TAY SACHS Protocol and wait for the protocol to complete before creating a new TAY SACHS protocol for the selected cartridge. If Yes is selected, the pad usage for the new protocol may not map correctly.

Note: A maximum of six TAY SACHS protocols may be run on a NanoChip 400 Cartridge. After all test sites on the cartridge have been used once


of 26

02-01/2013 140959.TS

with the TAY SACHS protocol, the cartridge may be reused with the TAY SACHS protocol a maximum of five times.

Figure 1. TAY SACHS Window

6. Select Materials Configuration and enter sample names manually, or select Import Content to import sample information from a Plate Content Definition Microsoft® Office Excel template. You may also enter information into the Description box if desired.

Note: Be sure sample names entered correspond to the wells used in setting up the sample plate.

If the number of samples exceeds the available sample positions on the

cartridge, the software will notify the user.

7. Select the TAY SACHS step in the Protocol Structure tree, and select the wells in the 96-well plate display, which have a dot. Note that the number of wells equals the number of samples to be analyzed.

Notes: The sample dilution option must be set in the TAY SACHS template

in the Protocol Editor such that the PERFORM ONBOARD DILUTION setting

is checked for automated onboard dilution and unchecked for

manual dilution.


of 26

02-01/2013 140959.TS

Multiple wells may be selected simultaneously by clicking the row (A – H) or

the column (1 – 12) on the sample plate in the TAY SACHS template.

8. Select File/Save As from the command tool bar. Enter a name for the protocol and save it.

Running the Assay

1. From the Instrument Manager, select Open from the Manager Panel screen. Browse to

select the protocol file you just created.

2. Note: When running a re-use protocol a dialog box will appear warning the user that pads have been previously addressed and requires a password to continue. Scroll to the bottom of the dialog to obtain the necessary password

3. The system calculates the amount of waste the protocol will generate. Check the waste bottle, making sure it has enough room to hold the new waste. If there is room, select Continue. If the waste bottle does not have enough room, empty it before running the assay, and then select Continue.

4. Load reagents on the instrument deck

a. Place the following buffer bottles on the instrument deck as instructed by

the Instrument LCD prompts.

Table 5: Location of Bottles in the NanoChip 400

Instrument

Solution Bottle

Size

Location

High Salt Buffer 30 mL Slot 1

Low Salt Buffer 30 mL Slot 2

Target Prep Buffer 30 mL Slot 3

CAPdown Sample Buffer B* 30 mL Slot 4

*Required for Onboard Sample Dilution option only. This position is left

empty when sample dilution is done manually.


of 26

02-01/2013 140959.TS

b. Place the Reagent Pack Plate in Plate Location 1 of the instrument deck as instructed by the LCD prompt.

Reagent packs are loaded into a Reagent Pack Plate before they are placed in the instrument deck as follows:

Tay Sachs Cap/Rep Reagent Pack – Position 1

c. Place the sample plate in Plate Location 2 of the instrument deck as instructed by LCD prompt.

Notes: When using a Nunc 96 well sample plate on deck, always position the plate with

well A1 in the bottom right-hand corner.

When using an ABI 96-well sample plate on deck, always position the plate with

well A1 in the upper left-hand corner. 5. After the run is complete, select Eject from the Instrument Manager screen. When the

LCD displays “Remove Cartridge”, remove the cartridge from the instrument. If the cartridge has not been fully used, return the cartridge to its pouch and store at 2-8°C. If the cartridge has been fully used, discard it. Note: When the eject button is selected, a window will appear asking the user to strip and/or fill

the cartridge before ejecting. Select Fill scroll down and choose Water. 6. Remove all buffers and replace the Wash Buffer with water. Perform routine

maintenance as appropriate.


of 26

02-01/2013 140959.TS

Analyzing the Data

A detailed description of the assay format can be found in Appendix A. Briefly, the genotyping calls are based on a green-to-red ratio where green indicates the presence of the wild type allele, and red the presence of the mutant allele. The data are analyzed in a Microsoft Office Excel based spreadsheet. Refer to Appendix B for a description of the TAY SACHS Data Analysis Spreadsheet features, instructions for setting preferences, and data calculations. 1. Export the data from a TAY SACHS NanoChip 400 run as follows:

a. Select Data Analysis from the NanoChip 400 Dockbar.

b. Select Export Processed Data. Select Next.

c. Select the appropriate cartridge and session number. The session numbers are listed by date, followed by the time the assay run started.

d. Select all 4 green and all 4 red image data files; select Finish.

e. A new screen displays. In the View tab, select Show Non-Activated Pads.

f. Select Export on the lower right side of the NanoChip 400 Data Analysis window.

g. A new screen appears; be sure all the boxes are checked and select Export.

h. Enter a file name (for example, the cartridge serial number and date of the run) and select Save. An Excel spreadsheet is automatically generated.

i. Close the NanoChip 400 Data Analysis software.

2. Import the TAY SACHS data into the TAY SACHS Data Analysis Spreadsheet

a. Open the TAY SACHS Data Analysis Spreadsheet.

b. Select the Import button. Find the file you just saved and select Open.

c. A new message appears that prompts the user to save the Data Analysis Spreadsheet. A default name of “cartridge number_session number” is given, but another name may be assigned.

Notes: If Show Non-Activated Pads was not selected during data export, an error message will appear when data import is attempted to the TAY SACHS Data Analysis Spreadsheet. If this occurs, repeat the data export process with the Show Non-Activated Pads selected.

To prevent data overwriting, the Import button is removed after a set of data is imported.

d. Save your changes to the spreadsheet.


of 26

02-01/2013 140959.TS

Appendix A: TAY SACHS Assay Format

Assay Format

The TAY SACHS assay uses a capture down format to genotype the markers based on identified sample. Following the single tube multiplex polymerase chain reaction, the amplicons are specifically bound to a permeation layer that covers the electronic microarray via hybridization to complementary capture oligonucleotides. These capture oligonucleotides are biotinylated at the 5’ or 3’ end and are bound to streptavidin that has been incorporated into the permeation layer. The TAY SACHS Kit components include the following:

TAY SACHS Primer Mix: set of forward and reverse amplification primers that specifically amplify segments of the TAY SACHS genes. LS Amplification Buffer: a general-purpose reagent used for the PCR amplification of DNA in an ionic environment optimized for analysis on the NanoChip 400 electronic microarray.

TAY SACHS Cap/Rep Reagent Pack: a 10 well pack containing a set of 4 unique capture and 4 unique reporter mixes. Each capture is a biotinylated synthetic oligonucleotide complementary to one of the amplicons generated with the TAY SACHS primer mix. Each capture is present in one of the four capture mixes. Reporter mixes contain discriminators and universal reporters. Each discriminator contains a segment that is complementary to the wild type or mutant allele. Those with a wild type complement also contain a segment that is complementary to the “wild type” universal reporter that contains a green fluorophore. Those with a mutant complement also contain a segment that is complementary to the “mutant” universal reporter that contains a red fluorophore. Each reporter mix contains 1 to 2 pairs of discriminators. CAPdown Sample Buffer B: a general-purpose reagent used for the delivery of amplicons to the activated test sites on the NanoChip 400 electronic microarray.

Starting with the amplified material, the TAY SACHS protocols generated as described in the “Creating a Protocol” section consist of the following five distinct steps.

1. Capture addressing: the capture oligonucleotide mixes specific for the TAY SACHS assay are

electronically addressed to predetermined pads across the cartridge in a sequential manner. The number of pads addressed with each mix is equal to the number of samples/controls being analyzed. Well 2-5 of the TAY SACHS Cap/Rep Reagent Pack contain Capture mixes 1 to 4.

2. Amplicon Hybridization: amplification reaction products diluted in CAPdown Sample Buffer B are

simultaneously addressed to 4 pads that comprise the full set of the Capture Mixes 1-4. The amplicons are sorted across the 4 pads by hybridization to specific captures. An amplicon hybridizes to just 1 of the 4 capture pads.


of 26

02-01/2013 140959.TS

3. Reporting: sequential cycles of passive hybridization-thermal discrimination-fluorescence imaging-thermal stripping ensue for each of the 4 reporter mixes contained in the TAY SACHS Cap/Rep Reagent Pack. The thermal stripping step removes the discriminator/universal reporters but leaves the amplicon bound to the capture oligonucleotide for the next reporter mix. Well 6-9 of the TAY SACHS Cap/Rep Reagent Pack contain Reporter mixes 1 to 4.

4. Reverse Bias Washing: each pad that was addressed with sample is subjected to a reverse

biasing to remove bound amplicon that can potentially interfere with future assays on the microarray. After Reverse Bias Washing, the system automatically fills the cartridge with Water for storage between uses.

The following tables and figures map the capture, sample, and background pad locations to the 16 X 25 array of the NanoChip Cartridge. Numbers 1-96 in Table 6 refer to the sample position of sequentially addressed samples. Note that each sample is addressed to 4 pads in a 1 X 4 block.

Figure 2 maps the location of the capture mixes within the 4 pads of each sample. The TAY SACHS template automatically maps samples starting with the first available sample position: for the first use of a cartridge, the first sample is addressed to sample position 1; if 10 sample positions were used in the first TAY SACHS run, the second use will begin with sample position 11. The TAY SACHS template automatically maps the capture mixes to the sample positions that are being used in the current TAY SACHS run. The sample position in the TAY SACHS Data Analysis Spreadsheet is referenced in three locations: the Samples Worksheet, Summary Worksheet, and Data Table Worksheet.

Table 6 : Sample Position

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

1 1 1 1 1 33 33 33 33 65 65 65 65 2 2 2 2 34 34 34 34 66 66 66 66

2 17 17 17 17 49 49 49 49 81 81 81 81 18 18 18 18 50 50 50 50 82 82 82 82

3 9 9 9 9 41 41 41 41 73 73 73 73 10 10 10 10 42 42 42 42 74 74 74 74

4 25 25 25 25 57 57 57 57 89 89 89 89 26 26 26 26 58 58 58 58 90 90 90 90

5 5 5 5 5 37 37 37 37 69 69 69 69 6 6 6 6 38 38 38 38 70 70 70 70

6 21 21 21 21 53 53 53 53 85 85 85 85 BG 22 22 22 22 54 54 54 54 86 86 86 86

7 13 13 13 13 45 45 45 45 77 77 77 77 14 14 14 14 46 46 46 46 78 78 78 78

8 29 29 29 29 61 61 61 61 93 93 93 93 30 30 30 30 62 62 62 62 94 94 94 94

9 3 3 3 3 35 35 35 35 67 67 67 67 4 4 4 4 36 36 36 36 68 68 68 68

10 19 19 19 19 51 51 51 51 83 83 83 83 20 20 20 20 52 52 52 52 84 84 84 84

11 11 11 11 11 43 43 43 43 75 75 75 75 BG 12 12 12 12 44 44 44 44 76 76 76 76

12 27 27 27 27 59 59 59 59 91 91 91 91 28 28 28 28 60 60 60 60 92 92 92 92

13 7 7 7 7 39 39 39 39 71 71 71 71 8 8 8 8 40 40 40 40 72 72 72 72

14 23 23 23 23 55 55 55 55 87 87 87 87 24 24 24 24 56 56 56 56 88 88 88 88

15 15 15 15 15 47 47 47 47 79 79 79 79 16 16 16 16 48 48 48 48 80 80 80 80

16 31 31 31 31 63 63 63 63 95 95 95 95 32 32 32 32 64 64 64 64 96 96 96 96


of 26

02-01/2013 140959.TS

Figure 2: Map of Capture Mix Pads Within a Sample

Figure 3 displays the map of markers reported across the 4 reporting mixes. Each reporter mix reports markers across the 4 sample pads. The unused pad serves as the background for that reporting. Each sample has its own background pad. The pad used for the background in each reporting is designated “CONTROL” in the figure. For example, Reporter mix 3 reports R170Q G>A on capture pad 1. Pad 3 is marked CONTROL and used as the background pad in data calculations for the mutation 1 R170Q G>A (see details in Appendix B).

Pad 1 Pad 2 Pad 3 Pad 4

Capture Mix 1 Capture Mix 2 Capture Mix 3 Capture Mix 4

Reporter Mix 1 Control IVS12+1 G>C

Reporter Mix 2 IVS5-2 A>G Control

Reporter Mix 3 R170Q G>A Control

Reporter Mix 4 1278insTATC Control G269S F 304/305

Figure 3: Map of Reporter Mixes 1–4 Across Capture Pads 1–4

Capture mix 1

Capture Mix 2

Capture Mix 3

Capture Mix 4

1 1 1 1


of 26

02-01/2013 140959.TS

Appendix B: TAY SACHS Data Analysis Spreadsheet and Data Calculations

Getting Started The security and preferences for the Data Analysis Spreadsheet require setting the first time the sheet is used. Security Setting The TAY SACHS Data Analysis Spreadsheet is a Microsoft Excel Workbook; imported data are calculated to results and genotyping calls using a macro. The Excel security setting must be set to medium or low to allow the use of macros. To adjust the security setting, open Microsoft Excel and select Options from the Tools menu in Excel. Under the Security tab, select the Macros Security box and select Medium. Select Ok. Always select Enable Macros when prompted. Read Only The TAY SACHS Data Analysis Spreadsheet is a Read-Only file and will prompt the user to save the file with a new name when preferences are set. Preference Setting

1. Information Header

Open the TAY SACHS Data Analysis Spreadsheet. Enter information for the header where prompted on the Samples Worksheet. The information header will appear on every worksheet and on every printed page.

2. Save Settings

Select File/Save As and save your preferences with a new file name.


of 26

02-01/2013 140959.TS

TAY SACHS Worksheets Samples Worksheet

The sample ID, cartridge number, cartridge session number, operator ID and instrument ID are imported to the Samples Worksheet. The Sample IDs and Sample ethnicities may be edited on this sheet. Boxes for the information header and comments are provided. All other cells are protected and cannot be edited. A footer with lines for “Reviewed By” and “Approved By” is on the printed sheet. Summary Worksheet

This sheet provides an overview of the sample calls. Sample positions that were run in the current session, sample IDs, Sample ethnicities, and genotyping results are displayed in adjacent columns. The genotyping column indicates the genotype for each mutation. Presence of a heterozygous genotype is indicated by “HET”, a homozygous mutant genotype is indicated by “HOM” and a marker with no mutation detected is indicated by ”–“. Samples with low signal are designated as “LS” for each marker with a low signal and samples with a no call are designated “NC” for each marker with a no call. If a marker is not part of the selected disease panel for the sample indicated, it will be grayed out in the spreadsheet. In the case where at least one marker is designated as “LS”, the sample needs to be retested and a new purification should be done from the original sample. The Summary Worksheet also displays the information header, cartridge number, cartridge session number, and operator ID. When printed, a footer with lines for “Reviewed By” and Approved By” are provided. The print settings for this sheet are editable. All cells in this sheet are protected and cannot be edited. Data Table Worksheet

The information displayed in the Data Table sheet are sample ID, cartridge number, cartridge session number, operator ID, and the calculated data that are described below. The information displayed for each sample and mix are the TAY SACHS marker, Green signal, Red signal, Green control, Red Control, Scaled G:R, Genotyping Call. The Data Table Worksheet also displays the information header, cartridge number, cartridge session number, and operator ID. When printed, a footer with lines for “Reviewed By” and “Approved By” are provided. The print settings for this worksheet are editable. All cells in this sheet are protected and cannot be edited.


of 26

02-01/2013 140959.TS

Data Calculations and Genotyping

The genotype for a sample is determined by the Green-to-Red signal ratio for each of the applicable markers. The Green signal indicates the presence of the wild type allele while the Red signal indicates the presence of a mutant allele. In viewing the Data Table Worksheet, the first column indicates the sample position on the cartridge. The sample column lists the sample ID. The marker column lists the markers present in the mix. Each row of markers for a sample corresponds to capture mixes 1-4 in order. The Green and Red listed for the CONTROL are the raw signals for that pad. The CONTROL signal is the sample specific background. The Green and Red listed for the markers detected are the raw signals for the marker. G:R is the value of the Green column divided by the Red column. This value is multiplied by a built-in scaling factor specific for each marker designed to make the call criteria constant across markers. The Call is the genotyping result for each marker, and is determined based on the signal requirements and call criteria described in Table 7

A marker is homozygous wild type if the Green-to-Red ratio is > 5, heterozygous if the Green-to-Red ratio is or falls between 0.33 and 3, and homozygous mutant if the ratio is < 0.2. A no call of “NC” is designated if the Green-to-Red ratio falls between 0.2 and 0.33 or between 3 and 5. Samples must meet signal criteria of > 2000 above sample control and > 2 fold above the sample control. For example, only green must meet the signal criteria for a TAY SACHS marker that has a G:R scaled value of > 5. A low signal designation of “LS” is used to denote samples that do not meet signal criteria.

Table 7: Call Criteria And Signal Specifications TAY SACHS

Genotyping--Reporter Mixes 1 - 4

Signal above control pad > 2000

(Signal-control pad) / control signal > 2

Scaled G:R for no mutation detected > 5

Scaled G:R for HET designation 0.33 < G:R < 3

Scaled G:R for HOMOZ designation < 0.2

Scaled G:R indeterminate ranges 3 < G:R < 5; 0.2 < G:R < 0.33


of 26

02-01/2013 140959.TS

Appendix C: Legal Notices

Notice to Recipients about Licenses European patents covering this product have expired. These patents are counterparts of issued U. S. Patents, 5,981,178; 6,001,588; 5,776,677 and applications and foreign counterparts thereof owned by The Hospital of Sick Children, Toronto, Canada and the University of Michigan, Ann Arbor, MI, USA. Certain usages of the product described herein may be covered by Genetic Technologies Limited, United States Patent No. 5,612,179, applications and foreign counterparts thereof and by The Johns Hopkins University United States Patent No. 5,407,796. You are authorized to practice the methods covered by or claimed in the above patent, but such authorized use is strictly limited to practice of such methods for or with the use of the product or products described herein. Any other use or commercialization of such methods requires a license directly from Genetic Technologies Limited. Persons wishing information regarding Genetic Technologies Limited and Jhons Hopkins University licensing terms should write to: Genetic Technologies Limited, Attention: Licensing Department, 60-66 Hanover Street, Fitzroy, Victoria 3065, Australia, and; Johns Hopkins University, JH Technology, 100 North Charles Street, 5

th Floor, Baltimore, MD

21201, USA. Persons wishing information regarding The Hospital of Sick Children, Toronto, Canada and the University of Michigan should write to: The Hospital of Sick Children, Director of Industry Partnership and Commercialization, Corporate Ventures, 525 University Avenue, Suite 1030, Toronto, Ontario M5G 2L3, Canada, and; The Office of Technology Transfer, University of Michigan, 1600 Huron Parkway, 2

nd Floor, Ann Arbor, MI 48109-2590, USA.

PCR information Although patents covering the basic polymerase chain reaction (PCR) have expired, patents covering the use of certain enzymes and other uses of the PCR process owned by Hoffman-LaRoche and others remain in effect and may require a license. Purchase of this product does not include or provide a license with respect to these patents. Savyon Diagnostics Ltd. does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of this product is recommended for persons that either have the license to perform PCR or are not required to obtain a license. No license under the patents to use the PCR process is conveyed expressly or by implication to the purchaser by the purchase of this product. Nothing herein is to be construed as recommending any practice or any products in violation of any patent or in violation of any law or regulation.

Limited Product Warranty

Savyon Diagnostics Ltd. warrants that this product will meet the specifications stated above. If any component of

this product does not conform to these specifications, Savyon Diagnostics Ltd. will at its sole discretion, as its sole

and exclusive liability and as the users’ sole and exclusive remedy, replace the product at no charge or refund the

cost of the product; provided that notice of non-conformance is given to Savyon Diagnostics Ltd. , within sixty (60)

days of receipt of the product.

This warranty limits Savyon Diagnostics Ltd’s liability to the replacement of this product or refund of the cost of the

product. NO OTHER WARRANTIES OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING WITHOUT LIMITATION

IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE OR NON-


of 26

02-01/2013 140959.TS

INFRINGEMENT, ARE PROVIDED BY SAVYON DIAGNOSTICS LTD. Savyon Diagnostics Ltd. shall have no

liability for any direct, indirect, consequential or incidental damages arising out of the use, the results of use or the

inability to use this product and its components.

In no event shall Savyon Diagnostics Ltd. be liable for claims for any other damages, whether direct, incidental,

foreseeable, consequential, or special (including but not limited to loss of use, revenue or profit), whether based

upon warranty, contract, tort (including negligence) or strict liability arising in connection with the sale or use or the

failure of Savyon Diagnostics Ltd. products to perform in accordance with the stated specifications.

Some components of nucleic acid analysis, such as specific methods and compositions for manipulating or

visualizing nucleic acids for analysis, may be covered by one or more patents owned by other parties. Similarly,

nucleic acids containing specific nucleotides sequences may be patented. Making, using, offering for sale, or selling

such components or nucleic acids may require one or more licenses. Nothing in this document should be construed

as an authorization or implicit license to make, use or sell any so covered component or nucleic acid under any

such patents.

Registered Trademarks

GeneAmp® is a registered trademark of Applied Biosystems.

Microsoft® is a registered trademark of Microsoft Corporation

Mastercycler® is a registered trademark of Eppendorf-Netheler-Hinz GmbH.

NanoChip® is a registered trademark of Gamida for Life B.V., The Netherlands.

Triton® is a registered trademark of Union Carbide Chemicals and Plastics Co., Inc.

MicroAmp™ is a registered trademark of Applera Corporation or its subsidiaries in the US and/or certain other countries.

REFERENCES

1.Kaback M, Lim-Steele J, Dabholkar K, Brown D, Levy N, Zeiger K. Tay-Sachs disease Carrier screening, prenatal diagnosis, and molecular era. J Am Med Assoc 1993:270:2307-2315.

2. Sandhoff K, Conzelmann E, Neufeld EF, Kaback MM, Suzuki K The metabolic basis of inherited disease, 6th ed. McGraw-Hill, New

York 1989:1807-1842

tay sachs application note€¦ · tay sachs disease (tsd) is a genetic neurodegenerative lysosomal...

Documents