Improved Real Time PCR Applications using Novel Eppendorf Amplification Technologies. Andrés Jarrin Application Specialist Eppendorf AG

Transcription

1 Real Time PCR Improved Real Time PCR Applications using Novel Eppendorf Amplification Technologies Andrés Jarrin Application Specialist Eppendorf AG

2 Specific Challenges of TaqMan like qpcr Real time qpcr with cleavable signaling probes is a multiplex assay by design, even with a single target Target Amplification (PCR) Enzymatic Signal Release (Probe Cleavage) Physical Signal Release & Detection (Excitation, Emission, & Fluorescence Detection) Signal Amplification All 3 processes run simultaneaously, but have diffrerent optimal reaction conditions. Challenge: - To find the most efficient compromise.

3 Specific Challenges of TaqMan like qpcr Approach to improve qpcr results: Novel Hot Start Technology Optimize Buffer Systems

4 The HotMaster Inhibitor Technology DNA Substrate Front View Front View Competitive Affinity Ligand ph Monovalent Ion Concentration Inhibitor Conc. Taq DNA Pol. Conc. Temperature 60 C 30 C

5 The HotMaster Inhibitor Technology Characteristics: Competitively Binding Affinity Ligand Mimiking the DNA Substrate of Polymerases Inhibitor Binding and Dissociation Are Reversible and Temperature-dependent Provides Hot Start and Cold Stop (Brake): Permanent Control of Primer Extension Specificity throughout PCR cycling Mode of Action: Retardation of the DNA Polymerase below Permissive Temperature (60 C) through Reduction of the Extension Rate and Processivity No Heat-activation of the Polymerase Necessary Universal Inhibitors for Thermophilic DNA Polymerases Inert to Intercalating Fluorescent Dyes used for Detection No Interaction with Primers and Template DNA

6 The HotMaster Inhibitor Technology 5 Minutes Primer Extension with 2 U Taq DNA Polymerase with & without HotMaster Inhibitor Temperature in C SS Fully ds M13 DNA (C) Intermediate Primer Extension Products (B) ss M13 Template DNA (A) A B C ss M13 Template DNA (A) Intermediate Primer Extension Products (B) Fully ds M13 DNA (C)

7 Hot Start - Cold Stop Feature ds Full Extension Product ss Template Extension Assay on primed ssm13 DNA with Hotstart Polymerases Before and After Heat Treatment (10 cycles) ds Full Extension Product After 10 PCR Cycles ss Template Temperature 37 C 50 C 60 C Hot Start Technique AB HM AB HM AB HM Inhibition with Cycling + +/- Inhibition without Cycling /- +

8 The HotMaster Inhibitor Technology Amplification of a 2 kb ß-globin target on human genomic DNA Controls HotMaster Conc. M M Taq HotM A B C Amplification of a 131 bp TNFα target on human genomic DNA Standard Taq HotMaster Taq A B

9 Specific Challenges of TaqMan like qpcr Approach to improve qpcr results: Novel Hot Start Technology Improved Optimize Buffer Systems

10 Influence of the Buffer on C t C A B A A C B C B C ph <8.2 <8.2 < <8.2 <8.2 <8.2 < K-Glutatmate * * * * Zwitterionic * * * * * * RealMaster Buffer

11 Influence of the Buffer on Signal and RFU C A B A A C B C B C ph <8.2 <8.2 < <8.2 <8.2 <8.2 < K-Glutatmate * * * * Zwitterionic * * * * * * Buffer *RealMaster

12 Primer Dimer Supression by Non-canonical dntps

13 Primer Dimer Supression by Non-canonical dntps

14 Reproducibility Across 96 Wells 2.8 Cycle Ct Shift Eppendorf RealMasterMix Probe ROX Competitor 2.8 Cycle Ct Shift Eppendorf RealMasterMix Probe ROX Competitor A

15 Storage Stability at Various Temperatures Eppendorf RealMasterMix 6 weeks +4 o C 6 weeks -20 o C 6 weeks -80 o C Competitor s Master Mix: 6 weeks +4 o C 6 weeks -20 o C 6 weeks -80 o C Six Week Storage Stability Assay Target: B2M Detector: FAM Machine: BioRad IQ icycler +4oC -20oC -80oC Average C t Avg Ct: Std Dev: 0.06 N: 3 Avg Ct: Std Dev: 0.06 N: Avg Ct: Std Dev: 0.06 N: 3 Avg Ct: 24.7 Std Dev: 0.10 N: 3 Avg Ct: Std Dev: 0.15 N: 3 Avg Ct: Std Dev: 0.38 N: 3 23 Eppendorf RealMasterMix Probe Competitor s qpcr master mix

16 Freeze/Thaw Storage Stability Cycle Freeze-Thaw Stability Assay : Average C t Value (4 Replicates Per Cycle) Average Ct Value across 15 freeze-thaw cycles Average Ct Value EPPENDORF RealMasterMix Probe BioRad iq icycler EPPENDORF RealMasterMix Probe ROX ABI 7000 SDS

17 Thanks to: R&D, Boulder, Colorado: Ryan Westberry Jessica Goodrich Melissa Garner Allan Roberts Eppendorf AG, Hamburg Andreas Gigler Andrés Jarrin Product Applications Wilhelm Pluester JaNae Grutt Jennifer Halcome Gerry Huitt