Thermal cycler amplification robustness: a comparison of several models

Transcription

1 APPLICATION NOTE Thermal cyclers Thermal cycler amplification robustness: a comparison of several models Introduction The ability of a thermal cycler to amplify difficult targets is a critical factor for many researchers. Ideally, a thermal cycler should produce high yields for a variety of difficult-to-amplify targets, including GC-rich and long templates. This study compares the amplification robustness of several thermal cyclers by measuring the absolute yield of a selection of difficult-to-amplify templates. Materials and methods The instruments tested in this study are shown in Table 1. The same equipment, methods, and reagents were used for each instrument. Table 1. s tested for amplification robustness. Manufacturer Model name Cat. No. Thermo Fisher Scientific ProFlex 96-Well PCR System Thermo Fisher Scientific SimpliAmp Thermal A24811 Bioer GeneMax Thermal BYQ6067 Bio-Rad C1000 Touch Thermal with 96-Well Fast Reaction Module Bio-Rad T100 Thermal Mastercycler Nexus Gradient Mastercycler Pro S Labcycler Gradient , Dice Touch TP350

2 AT-rich template (28.2% GC content) A single bulk reaction was prepared using AmpliTaq Gold 360 Master Mix (Cat. No ) according to the standard protocol along with primers targeting the RB1 oncogene. The forward primer (5 -TGTAAAACGACGGCCAGTCCTTAGGTGGATCAGCTGGGTG-3 ) and reverse primer (5 -CAGGAAACAGCTATGACCTCAATGAATTTGCATTTGTTGCAT-3 ) were used at 0.2 μm, and Human Genomic DNA (Roche, Cat. No ) template was used at 1 ng/μl. The bulk reaction was split among 32 wells to test for amplification consistency. The thermal cycling protocol consisted of a primary stage at 94 C for 10 minutes followed by a secondary stage consisting of 32 cycles at 95 C for 30 seconds, 60 C for 30 seconds, and 72 C for 30 seconds before a final stage at 72 C for 7 minutes. GC-rich template (80.4% GC content) A single bulk reaction was prepared using AmpliTaq Gold 360 Master Mix along with 360 GC Enhancer reagent and primers targeting the CCNE (cyclin-e1) gene. The forward primer (5 -TGTAAAACGACGGCCAGTAGCCAGGAAGATGGCCGATG-3 ) and reverse primer (5 -CAGGAAACAGCTATGACCTTCCGCGCGCAGGGATTT-3 ) were used at 0.2 μm, and Human Genomic DNA template was used at 1 ng/μl. The bulk reaction was split among 32 wells to test for amplification consistency. The thermal protocol was the same as that used for the AT-rich template. Dual-target amplification A single bulk reaction was prepared using AmpliTaq Gold 360 Master Mix according to the standard protocol along with primers that produce a high molecular weight (HMW) product and low molecular weight (LMW) product. The forward primer (5 -TGTAAAACGACGGCCAGTTCTCTTGCCTCAGCCTCCCA-3 ) and reverse primer (5 -CAGGAAACAGCTATGACCTTAAGCTGTTTCCATAGGGTGACACA-3 ) were used at 0.2 μm, and Human Genomic DNA template was used at 1 ng/μl. The bulk reaction was split among 32 wells to test for amplification consistency. The thermal protocol was the same as that used for the AT-rich template. Long template (12,342 bp) A single bulk reaction was prepared using the SequalPrep Long PCR Kit (Cat. No. A10498) according to the standard protocol along with primers targeting the BRCA2 gene. The forward primer (5 -CTCCCCCACAAAAAGGGGACAAAGC-3 ) and reverse primer (5 -ACAAACTCCCACATACCACTGGGGG-3 ) were used at 2 μm, and Human Genomic DNA was used at 2.5 ng/μl. The bulk reaction was split among 32 wells to test for amplification consistency. The thermal protocol consisted of a primary stage at 94 C for 2 minutes followed by a secondary stage consisting of 35 cycles at 94 C for 10 seconds, 61 C for 30 seconds, and 68 C for 14 minutes and 20 seconds before a final stage at 72 C for 5 minutes. Data acquisition and analysis After amplification, long PCR products were loaded onto an Invitrogen E-Gel 48 1% Agarose Gel (Cat. No. G800801). All other assays were loaded onto an Invitrogen E-Gel 48 2% Agarose Gel (Cat. No. G800802). The reactions were subjected to electrophoresis under standard conditions along with Invitrogen 100 bp DNA Ladder (Cat. No ), and gel images were acquired using a constant exposure time and aperture size. After imaging, bands were quantified using Thermo Scientific myimageanalysis Software (Cat. No ). Results Figure 1 and Figure 2 show the average yield produced from amplification of the AT-rich and GC-rich templates. Figure 3 and Figure 4 show the average yield produced from the two products amplified in the dual-target assay. Finally, Figure 5 shows the average yield produced from long-range amplification of the BRCA2 gene. The total number of wells that failed to amplify in each assay is summarized in Table 2.

3 12 AT-rich template PCR System Thermal Thermal Thermal Thermal Nexus Gradient Pro S Gradient Touch Avg. mass (ng) Figure 1. Average yield from amplification of the AT-rich template GC-rich template PCR System Thermal Thermal Thermal Thermal Nexus Gradient Pro S Gradient Touch Avg. mass (ng) Figure 2. Average yield from amplification of the GC-rich template Dual-target amplification, HMW product PCR System Thermal Thermal Thermal Thermal Nexus Gradient Pro S Gradient Touch Avg. mass (ng) Figure 3. Average yield of the HMW PCR product from dual-target amplification.

4 14.0 Dual-target amplification, LMW product PCR System Thermal Thermal Thermal Thermal Nexus Gradient Pro S Gradient Touch Avg. mass (ng) Figure 4. Average yield of the LMW PCR product from dual-target amplification Long template PCR System Thermal Thermal Thermal Thermal Nexus Gradient Pro S Gradient Touch Avg. mass (ng) Figure 5. Average yield from long-range amplification of the BRCA2 gene. Table 2. Summary of the total number of wells that failed to produce bands. Assay AT-rich template GC-rich template Dual target, HMW Dual target, LMW Long template (BRCA2) Applied Biosystems ProFlex 96-Well PCR System Applied Biosystems SimpliAmp Thermal Bioer GeneMax Thermal Bio-Rad C1000 Touch Thermal Bio-Rad T100 Thermal Mastercycler Nexus Gradient Mastercycler Pro S Labcycler Gradient Dice Touch

5 Discussion The ability of a thermal cycler to robustly amplify each reaction is pivotal to the user. In this study, we have shown that robustness of PCR amplifi cation varies between thermal cyclers. This was carried out as a side-by-side comparison while utilizing the same reaction chemistry. For best results, we recommend using a thermal cycler that produces a high yield for diffi cult-to-amplify targets but also does not sacrifi ce integrity and consistency of amplifi cation. The combined results for each assay suggest that the brand of thermal cyclers meet these criteria. Appendix ProFlex PCR System SimpliAmp Thermal Bioer GeneMax Thermal Bio-Rad C1000 Touch Thermal Bio-Rad T100 Thermal Mastercycler Nexus Mastercycler Pro S Labcycler Gradient Dice Touch Figure 6. Gel images of PCR products following amplification of AT-rich template.

6 ProFlex PCR System SimpliAmp Thermal Bioer GeneMax Thermal Bio-Rad C1000 Touch Thermal Bio-Rad T100 Thermal Mastercycler Nexus Mastercycler Pro S Labcycler Gradient Dice Touch Figure 7. Gel images of PCR products following amplification of GC-rich template.

7 ProFlex PCR System SimpliAmp Thermal Bioer GeneMax Thermal Bio-Rad C1000 Touch Thermal Bio-Rad T100 Thermal Mastercycler Nexus Mastercycler Pro S Labcycler Gradient Dice Touch Figure 8. Gel images of dimer PCR products.

8 ProFlex PCR System SimpliAmp Thermal Bioer GeneMax Thermal Bio-Rad C1000 Touch Thermal Bio-Rad T100 Thermal Mastercycler Nexus Mastercycler Pro S Labcycler Gradient Dice Touch Figure 9. Gel images of long PCR products following amplification of the BRCA2 gene.

9 Figure 10. Gel layout and plate coordinates of 2% E-Gel 48 Agarose Gel for amplification of GC-rich template, AT-rich template, and dual target (dimer). DNA refers to 100 bp DNA Ladder; TE refers to TE buffer. Figure 11. Gel layout and plate coordinates of 1% E-Gel 48 Agarose Gel for long-range amplification of BRCA2 template. DNA refers to 100 bp DNA Ladder; TE refers to TE buffer. Find out more at thermofisher.com/thermalcyclers For Research Use Only. Not for use in diagnostic procedures Thermo Fisher Scientifi c Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientifi c and its subsidiaries unless otherwise specifi ed. AmpliTaq Gold is a trademark of Roche Molecular Systems, Inc. GeneMax is a trademark of Bioer. T100 and C1000 Touch are trademarks of Bio-Rad Laboratories, Inc. Mastercycler is a trademark of AG. Dice is a trademark of Bio, Inc. CO