AJP 12 Application Note - Savyon Diagnostics · Glycogen Storage Disease Type 1a (GSD1a/ Von Gierke...

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AJP 12 Application Note (For use with NanoChip ® 400 Instrument)

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]

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

AJP 12 Application Note of 33

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Table of Contents

Introduction .........................................................................................3 Intended Use ................................................................................................ 3 Background .................................................................................................. 3 Kit Contents.................................................................................................. 6 Storage ....................................................................................................... 6 Using NanoChip Cartridges................................................................................ 7

NanoChip Cartridge Handling ....................................................................... 7

Materials and Equipment..........................................................................7 Materials Available from Savyon .......................................................................... 7 Additional Materials Available from Savyon ............................................................. 8 Other Required Materials (not available from Savyon)................................................. 8 Required Equipment........................................................................................ 8

Technical Assistance ...............................................................................9 Precautions........................................................................................ 10 Performing Sample Amplification.............................................................. 11 Extraction ................................................................................................... 11 Amplification ................................................................................................ 11 Preparing the Sample Plate .............................................................................. 12

Operating the NanoChip 400 System .......................................................... 14 Preparing Solutions for Use in the NanoChip 400 Instrument........................................ 14 Preparing the NanoChip Cartridge and Instrument .................................................... 15 Creating a Protocol ........................................................................................ 16 Running the Assay......................................................................................... 19 Analyzing the Data......................................................................................... 21

Appendix A: AJP 12 Assay Format............................................................. 22 Appendix B: AJP 12 Data Analysis Spreadsheet and Data Calculations .................. 26 Appendix C: Legal Notices ..................................................................... 30 REFERENCES ................................................................................................ 31


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Introduction

Intended Use The AJP 12 Kit is used to detect and identify a panel of 12 genetic diseases common to the Ashkenazi Jewish population.

For professional use only

Background

Similar to other ethnic populations, the Ashkenazi Jewish population has a higher prevalence of certain genetic disorders. These diseases are inherited in an autosomal recessive pattern. Affected individuals have inherited two copies of the mutated gene, one from each parent.

The AJP 12 assay was designed to diagnose the following diseases:

Alpha 1- Antitrypsin (AAT ) deficiency is the most prevalent potentially lethal hereditary disease of Caucasians. It leads to jaundice in infants, liver disease in children and adults, and pulmonary emphysema in adults. AAT is a protease inhibitor (PI), which protects tissue structures from damage by degrading enzymes. The genetic defect in AAT deficiency results in a molecule that cannot be released from its production site in hepatocytes. Low serum levels of AAT result in low alveolar concentration, where the protein normally would serve as protection against proteases. Consequential protease excess destroys alveolar walls and causes obstructive lung disease. Moreover, unsecreted AAT self-aggregates in the liver and causes liver disease. Mutations in the PI locus, located on chromosome 14, are associated with AAT deficiency. The most common risk alleles are PiS whose worldwide carrier rate is 1:50 (1:9 to 1:12 in Caucasians) and PiZ, with a worldwide carrier rate of 1:162 (1:30 to 1:40 in Caucasians) (8-13). Bloom Syndrome is inherited in an autosomal recessive fashion. Bloom Syndrome patients are much smaller than average, and often have a high-pitched voice and characteristic facial features including a long, narrow face, small lower jaw, a prominent nose and ears. They tend to develop pigmentation changes and dilated blood vessels in the skin. Other features of the disorder may include learning disabilities, mental retardation, chronic lung problems and diabetes. Men with Bloom Syndrome usually do not produce sperm while women with the disorder generally experience menopause earlier than usual. Chromosomal instability in Bloom Syndrome results in a high risk of cancer in affected individuals. Mutations in the BLM gene (locus 15q26.1) cause Bloom Syndrome. The BLM gene provides instructions for producing a protein called the Bloom (BLM) Syndrome Protein, which is a member of the DNA helicase family The carrier frequency in individuals of Eastern European ancestry is about 1:100 (22-23). Fanconi Anemia (FA) is an autosomal recessive disease characterized by progressive bone marrow failure, congenital anomalies, aplastic anemia and cancer susceptibility. There are at least 8 complementation groups (A-H). Extensive analysis of the FA group C gene FANCC in western countries revealed that 10-15% of FA patients have mutations in this gene. FANCC was mapped to chromosome 9 .The most common mutation is IVS4+4 A to T, a splice mutation in intron 4 which has been found mainly in patients of Ashkenazi Jewish ancestry. Patients with IVS4 mutation have a severe phenotype in comparison to other FA patients. The carrier


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frequency of the defective gene is 1:80 and the disease frequency is 1:25,600 within the Ashkenazi Jewish population (20). Familial Dysautonomia (FD) , also know as “Riley-Day Syndrome” or “Hereditary Sensory Neuropathy Type III”, is an autosomal recessive disorder that affects the development and survival of sensory, sympathetic, and some parasympathetic neurons. Individuals with FD are affected with a variety of symptoms, which include decreased sensitivity to pain and temperature, cardiovascular instability, recurrent pneumonias, vomiting crises, and gastrointestinal dysfunction (1, 2, 3 and 4). The major haplotype of FD is associated with mutation (2507+6T>C) that affects the donor splice site of intron 20 of the IKBKAP gene (6, 7). FD disorder is primarily confined to individuals of Ashkenazi Jewish descent. The carrier frequency of the defective gene is 1:30 and the disease frequency is 1:3600 within the Ashkenazi Jewish population (5).

Usher Syndrome is an autosomal recessive disorder characterized by bilateral sensorineural deafness and progressive loss of vision due to retinitis pigmentosa. It is the most frequent cause of deafness and concurrent blindness with a prevalence of 1 in 16000 to 1 in 50,000. There are three clinical subtypes of Usher Syndrome, the most severe of which is Usher Type 1 that involves deafness at birth, progressive blindness and balance problems. One of the Usher syndrome genes, PCDH15, is located on the long arm of chromosome 10. The R245X mutation accounts for a large proportion of cases of the type 1 Usher syndrome in Ashkenazi Jews, with a carrier rate as high as 1/40 (14). Canavan Disease is an autosomal recessive disorder that causes progressive damage to nerve cells in the brain. This disease is one of a group of genetic disorders called leukodystrophies. They are characterized by degeneration of myelin in the phospholipid layer insulating the axon of a neuron. The gene is located on chromosome 17. Canavan disease is caused by a defective ASPA gene which is responsible for the production of the enzyme aspartoacylase. Decreased aspartoacylase activity prevents the normal breakdown of N-acetyl aspartate, and the lack of breakdown somehow interferes with growth of the myelin sheath of the nerve fibers in the brain. Although Canavan disease may occur in any ethnic group, it affects persons of Eastern European Jewish ancestry more frequently. About 1/40 individuals of Ashkenazi Jewish ancestry are carriers. (25-27). Glycogen Storage Disease Type 1a (GSD1a/ Von Gierke Disease ) is an autosomal recessive disorder with an incidence of 1 in 100,000 births. GSD type 1a is caused by deficiency in the activity of glucose-6-phosphatase (G6Pase), a key enzyme in glucose homeostasis. Individuals with GSD1a exhibit a wide range of clinical symptoms including growth retardation, hypoglycemia, hepatomegaly, kidney enlargement, hyperlipidemia, hyperuricemia, tendency to bleed, neutropenia, hepatic adenomas and renal failure. GSD1a is caused by mutations in the G6Pase gene, located on chromosome 17. R83C is the most common mutation among Caucasians (15-16). Maple Syrup Urine Disease (MSUD) is a rapidly fatal neurodegenerative disease. MSUD is an inborn error of metabolism, resulting from the defective activity of branched-chain α-ketoacid dehydrogenase. The BCKDHB gene is located on chromosome 6q14. The enzymatic defect, transmitted in an autosomal recessive manner, results in an inability to catabolize leucine, isoleucine and valine. MSUD has been described in all ethnic groups and has an estimated worldwide frequency of 1:185,000, however ~30% of families with MSUD are of Ashkenazi


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Jewish descent. The R183P mutation accounts for most cases of MSUD in Ashkenazi Jews with a carrier frequency of 1:113(17). Nemaline Myopathy (NM) is a slowly progressive or now-progressive neuromuscular disorder characterized by muscle weakness and the presence of rod-shaped structures (nemaline bodies/rods) in affected muscle fibers. The estimated incidence is 1:50,000 live births. NM is found to be caused by mutations in three different genes, of which the most common in Ashkenazi Jewish population is the Nebulin gene mapped to chromosome 2q21.1-2q22. The carrier frequency of this deletion in Ashkenazi Jewish population is 1:108 (18-19).

Niemann-Pick Disease type A and B (NPD) is an inborn error of sphingomyelin catabolism that results from the deficient activity of the lysosomal hydrolase, acid. Type A NPD is a severe neurodegenerative disorder of infancy which leads to death by three years of age, whereas Type B NPD has a later age at onset and most patients survive into adulthood. NPD is caused by mutations in the NPC1, NPC2, or SMPD1 gene. The most common mutations in Jewish Ashkenazi population are located to the SMPD1 gene mapped to the chromosomal region11p15.1 to 15.4. Carrier frequency of NPD in Ashkenazi Jewish population is 1:90 to 1:100. Disease frequency is between 1:20,000 and 1:30,000 (21) Mucolipidosis (ML4) is a group of metabolic disorders inherited in an autosomal recessive manner. In ML4 patients, abnormal amounts of carbohydrates and lipids accumulate in cells. The symptoms range from mild learning disabilities to severe mental retardation and skeletal deformities. ML4 is classified as a lysosomal storage disease. The gene responsible for ML IV MCOLN1 makes the protein mucolipin-1. Due to mutations in the gene, mucolipin-1 is missing or dysfunctional in people with ML 4. The gene is mapped to a chromosomal region 19p13.2-13.3. Carrier frequency of ML4 is 1:110 and 1:40,000 births (24) Joubert Syndrome 2 (JBTS2) is a genetically heterogeneous autosomal recessive disorder characterized by psychomotor retardation, hypotonia, ataxia, nystagmus, and oculomotor apraxia and variably associated with dysmorphism, as well as retinal and renal involvement (28). JBTS2 is caused by mutations in TMEM216, which encodes an uncharacterized tetraspan transmembrane protein. In Ashkenazi Jewish, a single G35T mutation in exon 4 of the TMEM216 gene, results in an arg73-to-leu (R73L) substitution was identified as a founder mutation with a carrier rate of 1 in 92 (29).


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Table 1: Mutations detected by the AJP 12 Kit.

Kit Contents The AJP 12 Kit contains enough amplification buffer and primer mix for 64 samples/controls and enough detection reagents for two detection runs. One to 64 samples/controls can be analyzed in a single detection run. Refer to product package insert for performance characteristics and additional storage information.

Storage

Disease Mutation Gene

AAT PIZ PI

BLOOM Bloom 6 bp del / 7 bp ins BLM

USHER USHT1-R245X PCDH15

ML4-Del[ex 1-7] ML4

ML4-IVS3-2A>G MCOLN1

NPA-L302P

NPA-R496L

NPA-fsP-330 NP

NPB-∆R608

SMPD1

CAN-693 C>A CAN

CAN-854 A>C ASPA

FD-R696P FD

FD-2507+ 6 T>C IKBKAP

GS GSD1A-R83C G6PC

Nemaline Nemaline 24D25P Nebulin

MSUD MSUD-R183P BCKDHB

FAC FAC-IVS4+4A>T FANCC

Joubert Syndrome 2 G35T TMEM216

REF 800372 800375

≤-20°C ≤-20°C


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Using NanoChip Cartridges The AJP 12 Kit is designed to analyze 64 samples on a NanoChip 400 Cartridge. After all test sites on the cartridge have been used once with the AJP 12 protocol, the cartridge may be reused with the AJP 12 protocol a maximum of two times.

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 Savyon

REF Description Contents

FA 800372 AJP 12 Kit 64 Samples

AJP 12 Amplification Reagents 1 x vial (480 µL) Primer Mix 2 x vial (850 µL) LS Amplification Buffer

AJP 12 Reagent Packs 2 x Capture Reagent Packs 2 x Reporter Reagent Packs

1 x bottle CAPdown Sample Buffer B

FB 800372 AJP 12 Kit 128 Samples

AJP 12 Amplification Reagents 2 x vial (480 µL) Primer Mix 4 x vial (850 µL) LS Amplification Buffer

AJP 12 Reagent Packs 1 x Capture Reagent Packs 2 x Reporter Reagent Packs

2 x bottle CAPdown Sample Buffer B

800375 AJP 12Cap/Rep Reagent Packs 2 x Capture Reagent Pack 2 x Reporter Reagent Pack


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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 6 x bottles (25 mL each)

800155 NC400 High Salt Buffer 6 x bottles (25 mL each)

800156 NC400 Target Prep Buffer 6 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)

• 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) • Amplification Plates or Tubes • 2µm filters (Nalgene 5660020)

Required Equipment • NanoChip 400 System • Thermal Cycler1


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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


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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.


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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 AJP 12 Primer Mix from the ≤ -20°C

freezer. Thaw at room temperature and vortex.

Note: The LS Amplification Buffer and the AJP 12 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: PCR1 Master Mix Guidelines

Component Volume (µL)

LS Amplification Buffer 14.4

AJP 12 Primer Mix 7.5

FastStart Taq DNA Polymerase 1.1

Total Master Mix Volume per Reaction 23.0

3. Add 23µL of the PCR Master Mix to each reaction well in the PCR plate. 4. Add 2 µL of template DNA to each reaction well.

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

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


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Template DNA must be around 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 cycler1. 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

65 1 minute 40

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.

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 AJP 12 template in the Protocol Editor such

that the PERFORM ONBOARD DILUTION setting is checked � for automated onboard dilution

1 The following models are recommended:

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


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and unchecked for manual dilution. The template default has the PERFORM ONBOARD DILUTION setting checked � for automated onboard dilution.

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 ABI PCR 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. Use of other plate types may cause damage to the instrument.


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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 Ins trument 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 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 µm 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).


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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 packs from the freezer and place at room temperature to thaw.

� AJP 12 Capture Reagent Pack � AJP 12 Reporter Reagent Pack Notes: These reagent packs must be used within 8 hours of thawing.

Because all items listed above are 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.


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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 1-64 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 AJP 12 icon from the available templates (see Figure 1).

Note: The AJP 12 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 AJP 12 Protocol and wait for the protocol to complete before creating a new AJP 12 protocol for the selected cartridge. If Yes is selected, the pad usage for the new protocol may not map correctly.

Note: A maximum of three AJP 12 protocols may be run on a NanoChip 400 Cartridge. After all test sites on the cartridge have been used once with


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the AJP 12 protocol, the cartridge may be reused with the AJP 12 protocol a maximum of two times.

Figure 1. AJP 12 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.


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7. Select the AJP 12 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 AJP 12 template in the Protocol Editor such that the PERFORM ONBOARD DILUTION setting is checked � for automated onboard dilution and unchecked for manual dilution.

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

or the column (1 – 12) on the sample plate in the AJP 12 template.

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


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Running the Assay

1. Insert the thawed reagent packs in the Reagent Pack Plate as follows: � AJP 12 Capture Pack – position 1 (left most position) � AJP 12 Reporter Pack – position 2 Notes: Check the reagent packs for bubbles before inserting into the plate. If

bubbles are present, gently tap the bottom of the reagent pack on the bench top several times. Ensure that the reagent packs are flush with the top of the Reagent Pack Plate. Improper seating of the reagent packs may cause probe tip damage.

2. From the Instrument Manager, select Open from the Manager Panel screen.

Browse to select the protocol file you just created.

Note: When running a re-use protocol a dialog box will appear warning the user that pads have been previously addressed and require 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.


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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:

AJP 12 Capture reagent pack – Position 1 AJP 12 Reporter reagent pack – Position 2

c. Place the sample plate in Plate Location 2 of the instrument deck as

instructed by LCD prompt. Notes : 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.


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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 AJP 12 Data Analysis Spreadsheet features, instructions for setting preferences, and data calculations. 1. Export the data from AJP 12 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 6 green and all 6 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 AJP 12 data into the AJP 12 Data Analysis Spreadsheet

a. Open the AJP 12 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 AJP 12 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. Select the diseases for each sample in the Samples tab of the spreadsheet. The diseases available for selection are abbreviated. The default selection indicates the full panel of 17 markers to be analyzed. Select Analyze to view your results. Save your changes to the spreadsheet.


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Appendix A: AJP 12 Assay Format Assay Format

The AJP 12 assay uses a capture down format to genotype the markers based on identified sample ethnicity. 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 AJP 12 Kit components include the following:

AJP 12 Primer Mix: set of forward and reverse amplification primers that specifically amplify segments of the AJP 12 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.

AJP 12 Capture Reagent Pack: a 10 well pack containing a set of 6 unique capture mixes. Each capture is a biotinylated synthetic oligonucleotide complementary to one of the amplicons generated with the AJP 12 primer mix. AJP 12 Reporter reagent pack: a 10 well pack containing 8 unique reporter 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 AJP 12 reporter mix contains 2 to 4 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 AJP 12 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 AJP 12 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. Wells 1–6 of the AJP 12 Captures Reagent Pack contain Capture Mixes 1–6.


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2. Amplicon Hybridization: amplification reaction products diluted in CAPdown Sample Buffer B are simultaneously addressed to 6 pads that comprise the full set of the Capture Mixes 1-6. The amplicons are sorted across the 6 pads by hybridization to specific captures. An amplicon hybridizes to just 1 of the 6 capture pads.

3. Reporting: sequential cycles of passive hybridization-thermal discrimination-

fluorescence imaging-thermal stripping ensue for each of the 8 reporter mixes contained in the AJP 12 Reporter 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.

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 solution for storage between uses.


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The following tables and figures map the capture, sample, and reference pad locations to the 16 X 25 array of the NanoChip Cartridge. Numbers 1-64 in Table 6 refer to the sample position of sequentially addressed samples. Note that each sample is addressed to 6 pads in a 2 X 3 block.

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 17 17 17 5 5 5 21 21 21 2 2 2 18 18 18 6 6 6 22 22 22

2 1 1 1 17 17 17 5 5 5 21 21 21 2 2 2 18 18 18 6 6 6 22 22 22

3 33 33 33 49 49 49 37 37 37 53 53 53 34 34 34 50 50 50 38 38 38 54 54 54

4 33 33 33 49 49 49 37 37 37 53 53 53 34 34 34 50 50 50 38 38 38 54 54 54

5 9 9 9 25 25 25 13 13 13 29 29 29 10 10 10 26 26 26 14 14 14 30 30 30

6 9 9 9 25 25 25 13 13 13 29 29 29 BG 10 10 10 26 26 26 14 14 14 30 30 30

7 41 41 41 57 57 57 45 45 45 61 61 61 42 42 42 58 58 58 46 46 46 62 62 62

8 41 41 41 57 57 57 45 45 45 61 61 61 42 42 42 58 58 58 46 46 46 62 62 62

9 3 3 3 19 19 19 7 7 7 23 23 23 4 4 4 20 20 20 8 8 8 24 24 24

10 3 3 3 19 19 19 7 7 7 23 23 23 4 4 4 20 20 20 8 8 8 24 24 24

11 35 35 35 51 51 51 39 39 39 55 55 55 BG 36 36 36 52 52 52 40 40 40 56 56 56

12 35 35 35 51 51 51 39 39 39 55 55 55 36 36 36 52 52 52 40 40 40 56 56 56

13 11 11 11 27 27 27 15 15 15 31 31 31 12 12 12 28 28 28 16 16 16 32 32 32

14 11 11 11 27 27 27 15 15 15 31 31 31 12 12 12 28 28 28 16 16 16 32 32 32

15 43 43 43 59 59 59 47 47 47 63 63 63 44 44 44 60 60 60 48 48 48 64 64 64

16 43 43 43 59 59 59 47 47 47 63 63 63 44 44 44 60 60 60 48 48 48 64 64 64

Figure 2 maps the location of the capture mixes within the 6 pads of each sample. The AJP 12 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 AJP 12 run, the second use will begin with sample position 11. The AJP 12 template automatically maps the capture mixes to the sample positions that are being used in the current AJP 12 run. The sample position in the AJP 12 Data Analysis Spreadsheet is referenced in three locations: the Samples Worksheet, Summary Worksheet, and Data Table Worksheet.

1 1 1 1 1 1

Capture Mix 1

Capture Mix 2

Capture Mix 3

Capture Mix 4

Capture Mix 5

Capture Mix 6

Figure 2: Map of Capture Mix Pads Within a Sample


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Figure 3 displays the map of markers reported across the 8 reporting mixes. Each reporter mix reports up to 4 markers across the 6 sample pads and has a minimum of one pad not used. 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 1 reports ML4-Del[ex 1-7] on capture pad 3 and CAN-854 A>C on pad 5. No markers are reported on capture pads 1, 4 or 6. Pad 2 is marked Control and used as the background pad in data calculations (see details in Appendix B).

Capture Mix 1 Capture Mix 2 Capture Mix 3 Capture Mix 4 Capture Mix 5 Capture Mix 6

Reporter 1 - Control ML4-Del[ex 1-7] - CAN-854 A>C -

Reporter 2 MSUD-R183P FD-R696P - - Control G218T

Reporter 3 Bloom 6 del/7 ins Control - CAN-693 C>A FD-2507+ 6 T>C GSD1A-R83C

Reporter 4 Control NPA-R496L NPB-DR608 Nemaline 24D25P - ML4-IVS3-2A>G

Reporter 5 - USHT1-R245X Control - FAC-IVS4+4A>T -

Reporter 6 - Control NPA-L302P - - -

Reporter 7 - Control - NPA-fsP-330 - -

Reporter 8 - - Control - - AAT-PIZ

Figure 3: Map of Reporter Mixes 1–8 across Capture Pads 1–6


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Appendix B: AJP 12 Data Analysis Spreadsheet and D ata Calculations

Getting Started The security and preferences for the AJP 12 Data Analysis Spreadsheet require setting the first time the sheet is used. Security Setting The AJP 12 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 AJP 12 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 AJP 12 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.


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AJP 12 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 AJP 12 marker, Green signal-Control, Red signal-Control, Scaled G:R, Genotyping Call, Green/Control, and Red/Control. 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.


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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 1-6 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 less the CONTROL pad. 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

Table 7: Call Criteria And Signal Specifications AJP12

Genotyping--Reporter Mixes 1 - 8 Signal above control pad > 1500 (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 HOM/HET designation 0.2 < G:R < 0.33 Scaled G:R for HOMOZ designation < 0.2

Scaled G:R indeterminate ranges 3 < G:R < 5

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 3 and 5. A “HOM/HET” is designated if the Green to Red ratio falls between 0.2 and 0.33. Samples must meet signal criteria of > 1500 above sample control and > 2 fold above the sample control. For example, only green must meet the signal criteria for a AJP12 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.


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Table 8 summarizes the actions required for failed references, and for NC and LS designations.

Table 8: Action for Failed Reference Criteria, NC and LS Designations

Description Criteria Action Failure reference signal

≤ 1500 above reference background ≤ 3 fold above reference background

In the event of a reference failure all samples on the cartridge require repeat testing; may use previously amplified samples

No Call (NC) designation

Scaled Green-to-Red ratio between 3 and 5 and green or red signal meet signal criteria

In the event of NC on a marker the sample in question requires repeat; requires re-amplification of sample

Low Signal (LS) designation (Sample)

≤ 1500 above sample control background ≤ 2 fold above sample background

In the event of a LS on any marker the sample in question requires repeat; requires re-amplification of sample


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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 have been licensed from Beckman-Coulter Incorporated under United States Patent No. 5,653,939 and foreign counterparts thereof, and may be covered by Genetic Technologies Limited, United States Patent No. 5,612,179, applications and foreign counterparts thereof as well as 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 Beckman-Coulter, Inc., Johns Hopkins University and Genetic Technologies Limited. Persons wishing information regarding Beckman-Coulter's, Inc, Genetic Technologies Limited and Johns Hopkins University licensing terms should write to: Beckman Coulter, Inc. 250 South Kraemer Boulevard, Brea, CA 92821-6232, USA; Genetic Technologies Limited, Attention: Licensing Department, 60-66 Hanover Street, Fitzroy, Victoria 3065, Australia, and; Johns Hopkins University, JH Technology, 100 North Charles Street, 5th 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, 1500 Huron Parkway, 2nd 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.


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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-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

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24. Sun M. Goldin E. Stahl. S. Falardeau J.L. Kennedy J.C. Acierno C.M. Colman M. Schiffmann R. and Slaugenhaupt S.A. Mucolipidosis type IV is caused by mutations in a gene encoding a novel transient receptor potential channel. Human Molecular Genetics,9:17 2471-2478 (2000).

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28. Edvardson, S., Shaag, A., Zenvirt, S., Erlich, Y., Hannon, G. J., Shanske, A. L., Gomori, J. M., Ekstein, J., Elpeleg, O. Joubert syndrome 2 (JBTS2) in Ashkenazi Jews is associated with a TMEM216 mutation. Am. J. Hum. Genet. 86: 93-97(2010)

29. Valente, E. M., Logan, C. V., Mougou-Zerelli, S., Lee, J. H., Silhavy, J. L., Brancati, F., Iannicelli, M., Travaglini, L., Romani, S., Illi, B., Adams, M., Szymanska, K., and 39 others. Mutations in TMEM216 perturb ciliogenesis and cause Joubert, Meckel and related syndromes. (Letter) Nature Genet. 42: 619-625 (2010)

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