APPLICATION INFORMATION

Transcription

1 -1895A APPLICATION INFORMATION Automated Solutions AUTOMATED METHODS FOR PCR SETUP ON THE IOMEK FX Lisa Knapp, Jason Fawcett, and Laura Pajak, Ph.D. eckman Coulter, Inc. Introduction Since the development of the PCR* amplification process, it has been applied to an expanding number of applications in medical, forensic, and basic research. Although the technology for PCR has improved over the years, problems occur due to human error during the manual handling of reagents and template DNA. Additionally, manual setup of PCR in a 96-well format is both tedious and time consuming; each manual transfer step has the potential to introduce another source of contamination. The methods for PCR setup described in this application bulletin address the increasing demand for high-throughput amplification reactions. As such, three automated methods for PCR setup, which vary with respect to primer and source DNA location, have been developed on the iomek FX. These methods also allow for more efficient use of time in the laboratory as the methods developed can prepare six to eleven 96-well plates in under 9 minutes. Materials and Methods PCR The plasmid DNA used was previously purified from E. coli JM105 containing puc18 using the Promega Wizard* SV 96 Miniprep kit on the iomek FX (1). A 1 µl aliquot of plasmid DNA (20-50 ng/µl) was amplified in a total reaction volume of 40 µl containing final concentrations of 1X SureStart* Taq reaction buffer, 0.25 mm dntps (Roche iochemicals, Cat. No ), 50 pmol primers (puc/m13 forward and reverse sequencing primers, Promega Cat. Nos. Q5601 and Q5421, respectively) and 0.3 µl SureStart Taq DNA Polymerase (Stratagene Cat. No ). The master mix and primer mix, if separate, were set up manually at room temperature and aliquoted into Greiner 96-well V-bottom plates (Greiner Cat. No ) for Methods 1 and 2 and 96-well U-bottom plates (Greiner Cat. No ) for Method 3. All reactions were set up at room temperature on a single arm iomek FX. The cycling parameters on the MJ Research, Inc., thermal cycler (Model PTC-100) were 94 C for 10 min. followed by 30 cycles of 94 C, 45 sec.; 52 C, 45 sec.; 72 C, 45 sec.; followed by a final extension for 20 min. at 72 C. Reactions were held at 4 C after completion. To evaluate the reaction, agarose gel electrophoresis was performed using 10 µl of the PCR sample. The samples were loaded onto a 2.0% E-Gel* (Invitrogen Cat. No. G ). A single reaction was also set up using a non-hot start Taq DNA polymerase (Roche iochemicals, Cat. No ). For this reaction, the initial denaturation step was reduced to 4 min. at 94 C.

2 Consumables To conserve reagents, 96-well V-bottom plates for Methods 1 and 2 (Greiner Cat. No ) or 96-well U-bottom plates for Method 3 (Greiner Cat. No ) were used for the master mix and primer solutions. The DNA source plates were 96-well U-bottom plates (Greiner Cat. No ). The PCR thermowell plates (eckman Coulter P/N ) were held in place by stacking on a 96- well U-bottom plate (Greiner Cat. No ). Foil lids (eckman Coulter P/N ) were manually placed on the PCR plates prior to thermal cycling. Results Method Development Three different methods were developed based upon the most frequently used configurations of starting reagents. Method 1 (Figure 1A) uses a master mix plate containing distilled H 2 O, 1X PCR buffer, dntps, and Taq. A separate primer plate contains both the forward and reverse primers. In this method, the template DNA is aliquoted from source plates and all components are then mixed in the PCR reaction plate. A throughput of six plates can be achieved with the deck setup as shown. Method 2 is similar to Method 1 with the exception of the amplification primers which are included in the master mix in Method 2, increasing the throughput to seven plates (Figure 1). Method 3 (Figure 1C) has the highest throughput of eleven plates due to the inclusion of primers with the master mix and prealiquoted source DNA. To allow for pipetting, an excess of 10% over the total volume of master mix and primer mix (if separate) required for the reactions should be made and aliquoted into the appropriate 96-well plates. The methods were developed with respect to maximizing throughput while minimizing the potential for cross contamination. Methods 1 and 2 aliquot the master mix into the PCR reaction plates first using the same tips for all transfers. Similarly, using a fresh box of tips, the primers are then transferred to the thermowell plates. To allow for the primer plate to be used for additional reactions, the amount of primer necessary for all transfers is aspirated in a single step and then dispensed to all the plates. Finally, the template DNA is transferred from the source plates using a fresh box of tips for each DNA source. In Method 3, the template DNA is pre-aliquoted into the PCR reaction plates. In order to minimize the potential for cross contamination, a new box of tips is used for each transfer of master mix to the reaction plates. Mixing of the reaction is performed after the final component is added to the PCR reaction plate. Each method was also assessed to minimize flyover of reagents by source DNA to reduce the threat of contamination. To achieve this, the DNA source plates are placed immediately next to the reaction plates to which they correspond. The order of addition of reagents for the three methods is shown schematically in Figure 2. To assess consistency within a method, three plates (first, middle, and last) were amplified using each method. In Figure 3, the electrophoresed products from the first, middle, and last plates of Method 1 are shown. Figure 4 shows the results from a single, representative plate from Methods 2 and 3. Slight variations in the yield of the products were seen between plates but no consistent pattern to these variations was observed, hence results of the experiments were not affected. NOTE: The methods developed and validated included 1 µl dispenses of template DNA, a pipetting volume that pushes the limits of the 200 µl head on the iomek FX. For maximum robustness, it is recommended that the DNA source material be diluted to a concentration which allows for a larger transfer volume. If larger DNA transfer volumes are used, the methods must be adjusted accordingly. Using the pipetting conditions in Methods 1 and 2, CVs of less than 5% have been obtained for the 1 µl DNA dispense. Assessment of Cross-Contamination Cross-contamination is a major concern for any PCR-based experiment. To examine intraplate contamination, wells within the DNA source plate were seeded with either puc18 plasmid DNA or distilled water (Figure 5A) and PCR was performed as described above. Wells outlined in black in Figure 5A were analyzed by gel electrophoresis for intraplate cross-contamination. No intraplate contamination was seen irrespective of the method (Figures 3 and 4). To examine interplate contamination, an experiment was designed using source DNA plates with different plasmids. The first source plate (X-CON) was seeded with puc-based plasmids bearing inserts of various sizes ( bp) (Figures 5 & 6A). The second source plate used was the puc18 plate used in the previously described experiments (Figure 5A). As such, the presence of an approximately 200 bp band in the amplification products for the X-CON plate would be indicative of interplate contamination. These plates were placed in positions DNA/PCR 1 and DNA/PCR 5 of Method 2, respectively (Figure 1). 2

3 C Figure 1. Deck setups used for the three PCR setup methods on the iomek FX. (A) Method 1 uses a master mix plate for the PCR reagents and a separate primer plate which contains both the forward and reverse primers. () Method 2 includes the forward and reverse primers in the master mix. (C) Method 3 requires the template DNA to be prealiquoted into the PCR reaction plates. 3

4 Figure 2. A schematic representation of the three methods shows the order of addition of reagents. 4

5 C Figure 3. A comparison between the (A) first, PCR 1, () middle, PCR 4, and (C) last, PCR 6 amplification reactions. All plates were prepared using a hot-start Taq polymerase with Method 1 as seen in Figure 1A. The lanes correspond to: Lane 1, φx174 DNA/HaeIII ladder + Lambda DNA/HindIII (1.0 µg each); Lanes 2-7, 10 µl of the amplification reaction from sample wells C5, C7, D6, E5, E7, and F6; Lanes 8-12, 10 µl of wells C6, D5, D7, E6 and F5 which were negative controls (see Figure 5A). 5

6 Figure 4. Representative agarose gels from PCR reactions prepared by (A) Method 2 and () Method 3 (see Figures 1 and C). The lanes correspond to: Lane 1, φx174 DNA/HaeIII ladder + Lambda DNA/HindIII (1.0 µg each); Lanes 2-7, 10 µl of PCR reaction mix from sample wells C5, C7, D6, E5, E7, and F6 which contained puc18 plasmid DNA; Lanes 8-12, 10 µl of PCR reaction mix from sample wells C6, D5, D7, E6 and F5 which were negative controls (see Figure 5A). 6

7 Figure 5. Within the PCR plates, (A) shaded wells were seeded with puc18 plasmid DNA. All other wells were seeded with distilled water. For the cross-contamination experiment, an additional () PCR plate (X-CON) was seeded with plasmids containing four different puc18 based β-lactamase inserts of 550 (p201), 700 (p176), 850 (p140) and 1000 bp (p116). The outlined areas were evaluated for cross-contamination by agarose gel electrophoresis. 7

8 C Figure 6. To analyze interplate cross contamination, Method 2 was run using the plate X-CON (Figure 5) in position DNA 1 and puc18 (Figure 5A) in position DNA 5. The (A) placement of the primers and the expected sizes of the amplification products are shown for the X-CON products. The results of the PCR reactions were analyzed for cross-contamination by agarose gel electrophoresis. The lanes () correspond to: Lane 1, φx174 DNA/HaeIII + Lambda DNA/HindIII (1.0 µg each); Lanes 2-7, 10 µl of C5, C7, D6, E5, E7, and F6 which contained puc18 plasmid DNA; Lanes 8-12, 10 µl of C6, D5, D7, E6 and F5 which were negative controls as shown in Figure 5A. The lanes (C) of the X-CON plate correspond to: Lane 1, φx174 DNA/HaeIII + Lambda DNA/HindIII (1.0 µg each); Lanes 2-12, wells 10 µl of C5, C6, C7, D5, D6, D7, E5, E6, E7, F5, and F6 as shown in Figure 5. The deck positions were chosen due to the potential of cross-contamination from flyover of used tips from PCR reaction plate 5 over PCR reaction plate 1. The wells analyzed were the same as those used in previous experiments (Figures 5A and ). No interplate cross contamination was observed by gel electrophoresis (Figures 6 and C). Taq Comparison PCR protocols using Taq DNA polymerase frequently recommend preparing the reactions on ice. Modified versions of Taq polymerase are available which enable preparation of samples at room temperature. These modified versions of Taq DNA polymerase employ a reversibly activated enzyme that uses an extended 94 C incubation for enzyme activation, thereby improving amplification specificity and reducing non-specific background amplification (2). As our methods are performed at room temperature, a comparison was done to assess any differences between regular and hot-start Taq polymerase. For the comparison, Taq DNA polymerase (Roche iochemicals, Cat. No ) was used for a PCR setup using Method 1. No significant difference in amplification or background was seen between the two types of Taq polymerase when evaluated by agarose gel electrophoresis (Figure 7). However, results may vary depending upon the conditions of an individual reaction. 8

9 Figure 7. A comparison of the amplification between (A) Taq DNA polymerase and () a hotstart Taq DNA polymerase when the reactions were set up at room temperature. oth amplification reactions were PCR reaction plate 1 using Method 1 as seen in Figure 1A. The results of the PCR reactions were analyzed by agarose gel electrophoresis. The lanes correspond to: Lane 1, φx174 DNA/HaeIII ladder + Lambda DNA/HindIII (1.0 µg each); Lanes 2-7, 10 µl of PCR reaction mix from sample wells C5, C7, D6, E5, E7, and F6 which contained puc18 plasmid DNA; Lanes 8-12, 10 µl of PCR reaction mix from sample wells C6, D5, D7, E6 and F5 which were negative controls (see Figure 5A). Conclusion The results of this application bulletin show that a single-arm iomek FX can efficiently set up PCR reactions in a 96-well format within the laboratory. An advantage of using the iomek FX to set up 96-well PCR reactions is that it requires a fraction of the time it would take to perform each transfer manually. Three different methods have been developed and validated based upon the most common configurations of starting reagents. Furthermore, the methods here have been shown to reliably produce the intended amplification product when setup is performed at room temperature. Since a major concern in PCR-based experiments is contamination, the methods were written to minimize the potential of cross-contamination by the prudent usage of tips and consideration to the order of addition of reagents. The experiments performed have shown that no cross-contamination occurred either within a single 96-well plate or between 96-well plates even in the absence of barrier tips. The methods developed for this application bulletin can be used in a variety of laboratory applications. In addition, they can be modified to suit the needs of a particular reaction with additional validation. References 1. A Wizard Approach to Generating High- Quality Plasmid DNA Using Promega SV 96 Miniprep Kits on the iomek FX. Application Information ulletin A-1893A, eckman Coulter, Inc. 2. SureStart* DNA Polymerase Instruction Manual. Stratagene Cloning Systems, LaJolla, California. Acknowledgements The authors wish to thank Curtis Farley for his technical assistance and Troy Cook, Paul Diaz, Raj Kurapati, Shun Luo, Monique Mason, and Graham Threadgill for critical readings of the manuscript. 9

10 * SureStart is a trademark of Strategene. E-Gel is a trademark of Ethrog iotechnologies Ltd. QIAprep is a registered trademark of QIAGEN Instruments. Wizard is a registered trademark of Promega Corp. PCR is covered by U.S. patents owned by Hoffmann- LaRoche, Inc. All other trademarks are the property of their respective owners. Developing innovative solutions in genetic analysis, drug discovery, and instrument systems. eckman Coulter, Inc N. Harbor oulevard, ox 3100 Fullerton, California Sales: Service: Telex: Fax: Worldwide ioresearch Division Offices: Australia (61) Canada (905) China (86) Eastern Europe, Middle East, Africa (41) France Germany (89) Hong Kong (852) / Italy Japan Mexico Netherlands Singapore (65) South Africa (27) /5 Spain (34) Sweden Switzerland Taiwan (886) U.K U.S.A eckman Coulter,Inc.