INFINITI MDR1 Assay

Application Notes

The steps identified in the Application Notes have been validated. Please follow these steps to ensure optimal results. No deviation from the Application Notes is allowed. Any change will have to be validated by AutoGenomics and approved/released via AutoGenomics Change Order.

For research use only (RUO). Not for use in diagnostic procedures.

AN-40103-H (CO 1753) (02/11) AutoGenomics, 2980 Scott St., Vista, CA 92081 Tel: 760 477 2248 www.autogenomics.com

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1.0

Assay Description The MDR1 Assay is designed to detect and identify the following allelic variants:

Gene -1G>A 61A>G MDR1

Polymorphism

1199G>A 1236C>T 2677G>T/A 3435C>T

The assay protocol includes the following five major processes: a) b) c) d) e) Multiplex PCR amplification of DNA. Fluorescent label incorporation using analyte specific primer extension (ASPE). Hybridization of the ASPE primers to a microarray followed by washing. Scanning of the microarray. Signal detection and analysis.

Steps (b) through (e) are performed using the INFINITI Analyzer. 2.0 Warnings and Precautions All specimens are potentially hazardous and care should be taken when handling materials of human origin. No test method can offer complete assurance that HCV, HIV or other infectious agents are absent. Follow the CLSI approved guideline (Molecular Diagnostics Methods for Infectious Diseases, MM03-A2). To minimize the risk of cross contamination, the sample preparation, PCR reaction set up and PCR product analysis should be performed according to the CLSI approved guideline (Molecular Diagnostic Methods for Genetic Diseases, MM01-A2). 3.0 Quality Control Maintain calibration of thermocycler according to manufacturers specifications. Maintain calibration of INFINITI Analyzer according to AutoGenomics specifications. 4.0 Reagent Storage Store the Reagents in accordance with package labeling. 5.0 Reagents and Kits (a) MDR1 BioFilmChip Magazine (Catalog # 03-109).
AN-40103-H (CO 1753) (02/11) AutoGenomics, 2980 Scott St., Vista, CA 92081 Tel: 760 477 2248 www.autogenomics.com

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(b) MDR1 Intellipac Reagent Module (Catalog # 03-209) containing: Reaction master mix: dNTPs Labeled-dCTP Allele Specific Primers Extension Reaction Buffer Hybridization Buffer (c) MDR1 Amplification Mix containing (Catalog # 03-309): dNTPs multiplex primer mix MgCl2 PCR reaction buffer (d) Wash Buffer (Catalog # 12-001) (e) Titanium Taq DNA Polymerase (Clontech, Cat# 639208) (f) Distilled water (DNAase and RNAase free) 6.0 Equipment and Consumables (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) 7.0 INFINITI PipetteTips (Catalog # 11-008) INFINITI Waste Tray Liners (Catalog # 11-002) INFINITI Waste Tray Stir Bars (Catalog # 11-006) INFINITI Temp Cycler Plate (Catalog # 11-005) 24 Well Plates / Lids (Catalog # 11-003) Pipettors Mini Centrifuge MicrofugeTube Racks Thermocycler Vortex 1.5mL Microcentrifuge Tubes

Specimen Requirements Purified DNA that is at a concentration of >25 ng/l and has a ratio of A260/280 >1.6.

8.0

Recommended DNA controls It is recommended that positive controls (heterozygous and/or homozygous variants) be included in each test run. In addition, a wild type (all reference alleles) control and a no template control (i.e., molecular grade water) should also be included in each test run. Coriell DNA samples (www.coriell.org) are suitable positive controls for many of the allelic variants. Please contact AutoGenomics for recommendations on use of Coriell DNA.

9.0

Amplification Reaction Note: Keep Titanium Taq DNA polymerase on ice. Note: Completely thaw reagents at room temperature.

AN-40103-H (CO 1753) (02/11) AutoGenomics, 2980 Scott St., Vista, CA 92081 Tel: 760 477 2248 www.autogenomics.com

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Note: Vortex the amplification mix tube for 2 to 5 seconds then centrifuge briefly to bring the contents to the bottom of the tube. Note: To avoid contamination, a separate area is recommended for assembly of the PCR reaction. Filter tips and gloves must be used when handling specimens and controls. 9.1 Prepare the PCR master mix. Amplification mix Titanium Taq polymerase Total volume of PCR Master mix 17.5 l 0.5 l 18.0 l

Note: Calculate the amount of each reagent needed based on the number of reactions. 9.2 Gently vortex the PCR master mix then dispense 18 l of master mix into wells of the 24-well plate. Add 2 l of sample DNA (25 ng/l) to each well. PCR master mix Sample DNA Total volume of amplification reaction 9.4 18.0 l 2.0 l 20.0 l

9.3

Place the 24-well plate, sealed with the provided silicon lid, in a thermocycler and immediately commence the amplification reaction using the following program. Step No. 1 2 Temperature C 94 94 64 - 58 (-0.5/cycle) 72 94 58 72 72 4 Time 2 min. 30 sec. 30 sec. 30 sec. 30 sec. 30 sec. 30 sec. 2 min Hold No. of Cycles n/a 12x

3 4

30x 1 n/a

Note: After each cycle in step 2b the temperature is decreased by 0.5C. When an Eppendorf Mastercycler EP was used with the ramp rate set at 75%, the total cycling time was 1 hour and 45 minutes (+ 5 min). If using other thermocycler models we recommend adjusting the ramp rate in order to obtain an equivalent total cycling time. 10.0 Loading the INFINITI Analyzer Load the assembled 24WP and lid, assay specific magazines, Intellipac, tips, and wash buffer into the INFINITI Analyzer. For operation of the INFINITI Analyzer, refer to the INFINITI Analyzer Operators Manual.
AN-40103-H (CO 1753) (02/11) AutoGenomics, 2980 Scott St., Vista, CA 92081 Tel: 760 477 2248 www.autogenomics.com

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