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Large-Scale PCR for Aptamer Screening |
Note: This data was first presented at BIO 2007 by researchers from Vandalia Research, Inc. and Marshall University. Please scroll to the bottom for full acknowledgements.
ABSTRACT
Aptamers are short DNA or RNA molecules which can bind specifically to small and large molecule targets. Aptamers can be identified by Systematic Evolution of Ligands by Exponential Amplification (SELEX) [1]. SELEX method exposes a random oligonucleotide library to a specific target. Nucleic acids that bind the target specifically are amplified and subjected to additional rounds of selection. Limitations to SELEX include:
- library size (typically only 1015 molecules can be synthesized in a library)
- mispriming of the random library with >5-10 PCR amplification cycles, resulting in truncated or extended products
- dropout of potentially strong aptamers that are not competitive in amplification
Recently, several researchers have developed non-SELEX methods of aptamer identification using capillary gel electrophoresis. However these methods are also limited by the quantity and randomness of the starting library material.
These barriers to aptamer identification are overcome using Vandalia Research Inc. Triathlon™ PCR amplification system. The Triathlon™’s continuous method for heating and cooling the PCR reagents facilitates processing liter scale volumes of PCR product without pooling products from multiple small tubes. This results in reduced labor, decreased turnaround time, and, most critically, a reduction in the number of opportunities for contamination. The Triathlon™ can be used to produce large volumes of product amplified for only a limited number of cycles which can then be concentrated for SELEX or non-SELEX screening.
We present here information on systematic optimization of PCR yield and quality of random 80mer oligonucleotide libraries for aptamer identification using the Triathlon™ PCR. Parameters varied include initial template concentration, Primer concentration, cycle number, enzyme concentration, MgCl2 concentration, PCR additives, cycle length and primer length. PCR yield is measured by OD260 and Agarose gel electrophoresis after Centricon cleanup of nucleotides and primers.
INTRODUCTION
Aptamers are identified by screening a random sequence library flanked with known PCR primers against a target molecule. Nucleic acids which bind the target are amplified and the process is repeated for several rounds of binding and amplification. Amplification of randomized oligonucleotide libraries is an essential step in aptamer identification. A library with at least 1014 to 1015 different sequences is desirable. A problem is that these libraries lose sequence diversity through multiple rounds of amplification and the products rapidly convert to longer by-products[2].
The objectives addressed in this project included the optimization of PCR of random oligonucleotide libraries with a standard Eppendorf Thermalcycler compared to the Triathlon™ at Vandalia Research to find the optimum parameters needed for maximum production of random aptamers. The factors that were optimized included: temperature, concentration of template, number of cycles and the effect of concentration on purity.
Oligonucleotide concentration was optimized first. This was done by diluting and aliquoting random libraries and determining the exact concentration in ng/µL spectrophotometrically. A range of concentrations were amplified by PCR and run on a gel to find the ideal concentration. Once this concentration was selected, cycle number was optimized by running individual samples at an increasing number of cycles. These were then run on a gel and the cycle number with the most correct amplicon was identified. Optimal PCR conditions were determined by performing a gradient of annealing temperatures. PCR products from gradients were run on gels and the best result was selected for further usage.
The best PCR products were obtained with 10 cycles at 94°C for 10 seconds, 65°C for 10 seconds and 72°C for 10 seconds. The concentration of the template that was selected was 3.8 X 107 pmol/ µL. This was chosen based on the amount of DNA usually used in finding aptamers as well as how much smearing the concentration caused. Smearing occurs more readily at higher concentrations. With a relatively low number of cycles and an optimum annealing temperature, the smearing was minimal.
MATERIALS AND METHODS
Chemicals and Materials - Taq DNA polymerase and buffer components and nucleotides were obtained from Takara (Kit). DNA library LAM and primers were made by IDT. All solutions were made with Milli-Q deionized water.
DNA Primers and Library - Two libraries of DNA product were used that could produce 80bp. The first, DNA library LAM, contained a central randomized sequence of 40 nucleotides flanked by 20 nucleotide constant regions. PCR amplification resulted in 80 bp long PCR products. The PCR primer set for this library (LAM) were 5’ –biotin-AAAGTTCTCGGGATCACCATC and 5’ GTTTCCGTTCTTCTTCGT. These primers were derived by using primer 3 on λ phage DNA. This library was HPLC purified and the random sequence was balanced by IDT to insure an even distribution of all 4 nucleotides within the sequence. The second library (LIC) was synthesized at the 1 µL scale and HPLC purified (Operon) as described by Murphy et al.[3]. This library composed of 40 nucleotide random sequences flanked by 20 nucleotide sequences suitable for ligation independent cloning (LIC) 5’-GGTATTGAGGGTCGCATC and 5’ AGAGGAGAGTTAGAGCCATC. Both libraries were diluted to 0.10 nM per µL in TE and stored at -80°C.
Conventional PCR - The PCR was performed and optimized on an Eppendorf Mastercycler Gradient Thermocycler. Reaction mix contained dNTPs, MgCl2, buffer, and varying concentration of DNA, and a proprietary additive. PCR was performed in 20 µL reaction mixes containing 1x Takara buffer, 1.5 mM MgCl2, 0.5 µM dNTP’s, 0.5 µM of each primer and DNA concentrations ranging from 1 µM to 10-14 µM. The PCR cycle used for LAM was optimized with no initial melt or final extension at 94°C for 10 s, 62 °C for 10 s, and 72 °C for 10 s. The number of cycles tested ranged from 5 to 35 cycles in 5 cycle increments. The cycle used for LIC also had no initial melt or final extension at 94 °C for 10 s, 58 °C for 10 s, and 62 °C for 10 s. This was also tested on a range of 5 to 35 in 5 cycle increments.
Large Scale Continuous PCR - The large scale PCR was performed using a Vandalia Research Triathlon™ instrument. Multiple 10 mL PCR reaction mixes containing concentrations of the reagents described previously with the template concentration at 10-2 µM were prepared and run for 5, 10 and 20 cycles. PCR mixes for the Triathlon™ also contained a proprietary additive. The Triathlon™ cycle was optimized without initial melt or final extension at 94°C for 10 s, 62°C for 10 s, and 72°C for 10 s.
PCR Product Analysis - PCR products were analyzed using 2% and 4% pre-stained, pre-cast, E-gels from Invitrogen. The gels were run for 15 to 30 minutes and imaged using Bio-Doc Imaging System (UVP.) PCR products were cleaned up from unincorporated nucleotides and primers using YM30 Microcon system (Millipore). DNA concentration was determined using a Nanodrop spectrophotometer.
RESULTS
Vandalia Research’s flagship technology is the Triathlon™ thermal cycler. The Triathlon™ is a patent-pending system invented to enable the mass production of specific, high-quality DNA sequences with the polymerase chain reaction (PCR). The Triathlon™ system will ultimately support throughput volumes of up to six liters of high-quality PCR product per day, suitable for large industrial applications. Vandalia Research specializes in large-scale DNA production services, using its proprietary Triathlon production system. Additionally, we provide stand-alone DNA products in retail and bulk quantities, such as DNA ladders and standards.
The best PCR results were obtained with 10 cycles at 94°C for 10 seconds, 65°C for 10 seconds and 72°C for 10 seconds. The optimal concentration of the template was 3.8 X 107 pmol/ µL. This was chosen based on the amount of DNA library variation required to find aptamers as well as the amount of byproducts produced. Byproducts cause smearing on the gels and occurs more readily at higher concentrations. With a relatively low number of cycles and an optimum annealing temperature, the smearing was minimal.
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Lane 1: Vandalia PCR Ladder; Lane 2,3:10-6 µM; Lane 4,5:10-8 µM; Lane 6,7:10-10 µM; Lane 8,9:10-12 µM; Lane 10,11:10-14 µM; Lane 12: Negative control |
Gel A: Lane 1: Vandalia PCR Ladder; Lane 2: Control (5 cycles); Lane 3: 5 cycles; Lane 4: 5 cycles; Lane 5: Control (10 cycles); Lane 6: 10 cycles; Lane 7: 10 cycles; 8: Control (15 cycles); Lane 9: 15 cycles; Lane 10: 15 cycles; Lane 11: Control (20 cycles); Lane 12: 20 cycles Gel B: Lane 1: Vandalia PCR Ladder; Lane 2: 20 cycles; Lane 3: Control (25 cycles); Lane 4: 25 cycles; Lane 5: 25 cycles; Lane 6: Control (30 cycles); Lane 7: 30 cycles; 8: 30 cycles; Lane 9: Control (35 cycles); Lane 10: 35 cycles (Concentration of template: 10-4µM) Gels C, D: follow the same pattern but have template concentrations of 10-2µM |
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Gel A: Lane 1: Vandalia PCR Ladder; Lane 2: Control (5 cycles); Lane 3: 5 cycles; Lane 4: 5 cycles; Lane 5: Control (10); Lane 6: 10 cycles; Lane 7: 10 cycles; 8: Control (15 cycles); Lane 9: 15 cycles; Lane 10: 15 cycles; Lane 11: Control (20 cycles); Lane 12: 20 cycles Gel B: Lane 1: Vandalia PCR Ladder; Lane 2: 20 cycles; Lane 3: Control (25 cycles); Lane 4: 25 cycles; Lane 5: 25 cycles; Lane 6: Control (30 cycles); Lane 7: 30 cycles; 8: 30 cycles; Lane 9: Control (35 cycles); Lane 10: 35 cycles; Lane 11: 35 cycles (Concentration of template: 10-2µM) |
Gel A: Lane 1: Vandalia PCR Ladder; Lane 2: Control (5 cycles); Lane 3: Control (10 cycles) ; Lane 4: Control (15 cycles); ; Lane 5: Control (20); Lane 6: Control (25 cycles); ; Lane 7: Control (30 cycles); ; 8: Control (35 cycles); Gel B: Lane 1: Vandalia PCR Ladder; Lane 2: 5 cycles; Lane 3: 10 cycles; Lane 4: 15 cycles; Lane 5: 20 cycles; Lane 6: 25 cycles; Lane 7: 30 cycles; 8: 35 cycles (Concentration of template: 10-2µM) |
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| Gel A. Lane 1: Vandalia PCR Ladder; Lane 2: Control (20 cycles on Eppendorf); Lane 3: 20 cycles (on Eppendorf); Lane 4:Omit; Lane 5: 20 cycles (Triathlon- 10µL of both water and product); Lane 6: 20 cycles (Triathlon- 15µL of water and 5µL of product); Lane 7: 20 cycles (Triathlon- 5µL of water and 15µL of product). Graph is yield in ng/ul in a 10 ml PCR reaction. |
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CONCLUSION
By optimizing the concentration of the library, the annealing temperature, and number of cycles for running PCR with the intention of selecting aptamers, a very effective system was created that can be used in future experiments. Larger pools of amplified oligonucleotides permit a more representative sample of random sequences to be screened for aptamers and facilitate development of new methods for aptamer identification. The Triathlon™ is also an effective production system for DNA aptamers.
AUTHORS
Elizabeth E. Murray (1,2), Ph.D. Michael L. Norton (1,2), Ph.D. Menashi Cohenford (2), Ph.D. Halima Al-Qawasmi (2), Nathan Gibson (2)
1 - Vandalia Research, Inc.
2 - Marshall University
REFERENCES & ACKNOWLEGEMENTS
1. Tuerk, C. & Gold, L. (1990) Science 249, 505-510
2. Musheev, MU, Krylov, SN. Selection of aptamers by systematic evolution of ligands by exponential enrichment: Addressing the polymerase chain issue. Analytica Chimica Acta. (2006); 564: 91-96.
3. Murphy MB, Fuller ST, Richardson PM, Doyle SA. An improved method for the in vitro evolution of aptamers and applications in protein detection and purification. Nucleic Acids Res. (2003);31(18):e110.
We would like to thank Ngoc Truong, Thao Nguyen, Valerie Patton, Steven Howard, and Marshall University for their assistance, and WV-EPSCoR and WV Space Grant Consortium for funding.
