Reliable PCR Components for Molecular Diagnostic Assays

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1 Reliable PCR Components for Molecular Diagnostic Assays Terri McDonnell, MBA, PMP Senior Program Manager, Molecular Diagnostics March 2014

2 In this webinar we will: Discuss requirements for amplification components used in PCR-based MDx assays Introduce new family of GoTaq MDx DNA Polymerases Describe Promega MDx PCR component product portfolio Discuss customization options for MDx PCR components The products discussed in this presentation are intended for general laboratory use and are suitable for use as components of molecular diagnostic assays, where applicable country laws allow. The products themselves do not provide any diagnostic result. 2

3 PCR in Molecular Diagnostic Applications Wide-spread Use Across Multiple Test Applications Key Molecular Test Applications and Technologies PCR is a common technology used across MDx segments Robust & Reliable MDx IVD PCR Assay Segment Microbiology/ Virology Histology Blood Screening Prenatal Inherited Diseases Tissue Typing Oncology Pharmacodiagnostics Most widely used technology PCR, bdna, TMA, sequencing FISH, PCR, microarray, sequencing PCR, TMA PCR, FISH, sequencing, cfdna Bead array, microarray, PCR, sequencing Bead array, PCR Bead array, microarray, PCR, sequencing, cfdna Bead array, PCR, sequencing Source: Kalorama Information. World Market for Molecular Diagnostics, January

4 PCR in Molecular Diagnostic Assays Consistency Depends on Quality Components dntps PCR Components Buffers MgCl 2 Enzymes Consistent Performance Utilize PCR components manufactured with: Production/Process Controls Equipment/Facilities Controls Material/Purchasing Controls Robust & Reliable MDx IVD PCR Assay 4

5 Quality Systems All Promega products are manufactured under our Quality System. Detailed manufacture and QC documentation Production and process controls Product Certificate of Analysis Traceable product batch records Product stability programs Product change control and notification procedures Supplier evaluation and qualification program Equipment maintenance and calibration program Non-conforming product procedures Qualified and trained personnel Design controls Scope of Quality System Controls Risk 5

6 Key Attributes for MDx PCR components Enzymes are a Critical Component dntps PCR Components Buffers MgCl 2 Enzymes Robust & Reliable Components MDx IVD PCR Assay PCR Engine Critical component Consistent unit activity Low contaminants Flexible formats 6

7 Introducing GMP-manufactured Taq DNA polymerases and hot start polymerases suitable for use in molecular in vitro diagnostic amplification assays. 7

8 Specifications High Quality and Consistent Performance GMP-manufactured Low DNA Contamination (bacterial, fungal, mammalian DNA) Undetectable Nuclease Contamination (exonuclease, endonuclease, nicking) Functionally Tested Multiple Formats Lot-specific Certificate of Analysis Customization Options Intended for Clinical Use- No License or royalties 8

9 Specifications Low DNA Contamination Low DNA Contamination (bacterial, fungal, mammalian DNA) Bacterial DNA Fungal DNA Mammalian DNA 1 unit contains < 10 copies bacterial gdna QC assay targets a conserved region of the 16S rrna gene 1 unit contains < 1 genome equivalent fungal gdna QC assay targets a conserved region of the 18S rrna gene 1 unit contains < 1 genome equivalent mammalian gdna QC assay targets a conserved region of mitochondrial DNA 9

10 Specifications Undetectable Nuclease Contamination Undetectable Nuclease Contamination Exonuclease No observable degradation of 1mg of Lambda DNA/Hind III Markers after incubation for 22 o C, followed by 45 o C in the presence of 15u Taq polymerase. Endonuclease Nicking No observable nicking of 0.5mg of supercoiled DNA after incubation for 22 o C, followed by 45 o C in the presence of 15u Taq polymerase. 10

11 Specifications Functionally Tested by Real-Time PCR Functionally Tested GoTaq MDx DNA Polymerase 1 unit of Taq polymerase quantitatively amplifies genomic DNA target meeting slope, y-intercept, and R 2 specifications. GoTaq MDx Hot Start Polymerase Amplify a hot-start model template to produce a single 1.5kb product, eliminating extraneous amplification products. In a non hot-start PCR, this template produces an additional 410bp amplification product. 11

12 Flexibility Stock and Custom Versions Available GoTaq MDx DNA Polymerase Multiple Product Formats GoTaq MDx Hot Start Polymerase 5 units/ml 5 units/ml 50 units/ml Lyophilization-ready, glycerol-free, formats Customizable 12

13 Flexibility Customization to Match Your Specific Requirements Customization Options Concentration Formulation Bulk Custom Dispensed Packaging / Labeling Not listed? Ask Us 13

14 GoTaq MDx DNA Polymerases Intended Use GMP-manufactured Taq DNA polymerases and hot start polymerases suitable for use in molecular in vitro diagnostic amplification assays. 14

15 Key Attributes for MDx PCR components dntps are a Required Component dntps PCR Components Buffers MgCl 2 Enzymes PCR Building Blocks Consistent concentration & purity Low contaminants Flexible formats Robust & Reliable Components MDx IVD PCR Assay 15

16 Specifications High Purity and Functionally Tested dntps Consistent Concentration Functionally Tested Purity Customization Options Catalog products: 100mM +/- 5mM Functionally tested in PCR HPLC: 99% 31 P NMR: 97% Triphosphate Tested for contaminating DNase and RNase activity Concentration Formulation Bulk Custom Dispensed Not listed? Ask Us Packaging / Labeling 16

17 Key attributes for MDx PCR components Buffer Systems are Key to Performance Optimization dntps PCR Components Buffers MgCl 2 Enzymes PCR Functional Environment Consistent formulations Low contaminants Flexible formats Robust & Reliable Components MDx IVD PCR Assay 17

18 Specifications MgCl 2 MgCl 2 Solution Consistent Concentration Functionally Tested Undetectable Contaminants Customization Options Endonuclease Activity Exonuclease Activity RNase Activity Concentration Custom Dispensed 25mM +/- 5mM Functionally tested in PCR No observable degradation or nicking after incubation with 1mg of plasmid DNA No observable degradation after incubation with 1mg of lambda DNA. No observable degradation after incubation with RNA. Not listed? Ask Us Bulk Packaging / Labeling 18

19 Specifications - Buffers PCR Buffers Catalog Standard end-point PCR Flexible Formulations Undetectable Contaminants Functionally Tested Customization Options Custom Endonuclease Activity Exonuclease Activity Custom Dispensed Real-time PCR Multiplex PCR GC-Rich Direct Sample Customer specified No observable degradation or nicking after incubation with 1mg of plasmid DNA No observable degradation after incubation with 1mg of lambda DNA Functionally tested in PCR Bulk Not listed? Ask Us Packaging / Labeling 19

20 Summary - Reduce Risk and Improve Performance with Quality Components dntps PCR Components Buffers MgCl 2 Enzymes Consistent Performance Utilize PCR components manufactured with: Production/Process Controls Equipment/Facilities Controls Material/Purchasing Controls Robust & Reliable Components MDx IVD PCR Assay 20

21 Product References: Please visit for more information. 21

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