Designing of a biochemical kit for low-cost real-time PCR. Lam Y.S. 1 and Lin Q.S. 2

Transcription

1 Designing of a biochemical kit for low-cost real-time PCR Lam Y.S. 1 and Lin Q.S. 2 Department of Biological Sciences, Faculty of Science, National University of Singapore 1 Kent Ridge Road, Singapore ABSTRACT Applied Biosystems (AB) is the sponsor of the Global Clinic project, which involves members of Harvey Mudd College (HMC) and the National University of Singapore (NUS). To further develop a low-cost Real-time PCR instrument for educational purposes built last year, the NUS Team has been tasked incorporate a compatible and robust biochemical protocol for the prototype. The duration of the PCR reaction is limited to a class period and should consist of only AB products at the lowest cost possible. All experiments were done on a StepOnePlus Real-Time PCR System from AB as a benchmark. DNA polymerase from Applied biosystems: AmpliTaq, AmpliTaq LD and SuperTaq are tested in this experiment for maximal efficiency. Bovine Serum Albumin (BSA), Tween 2 and 7-Deaza-2- deoxyguanosine 5 -triphosphate (dc 7 GTP) were tested as cosolvents which were reported to improve amplification and stabilize the enzyme. It was found that SuperTaq has the most efficiency. However, none of the cosolvents tested show significant improvement in amplification efficiency. Future works include testing a combination of cosolvents and altering the PCR reactions to mimic the slower rate of heating and cooling of the prototype. To test robustness, the effect of defrosting reagents on amplification efficiency would be tested. INTRODUCTION Applied Biosystems (AB) is the sponsor of the Global Clinic project, which involves members of Harvey Mudd College (HMC) and the National University of Singapore (NUS). The Global Clinic Team has developed a low cost real-time polymerase chain reaction (PCR) instrument for the educational market. To further develop this product, the Global Clinic Team has been tasked to optimize the prototype, incorporate a compatible and robust biochemical protocol and creating firmware with a user interface. NUS will focus on creating a robust biochemical protocol using components provided by AB that is compatible to the instrument. The duration of the PCR reaction is limited to the class period. The focus of the project is on educating high school students on PCR reactions and 1 Student 2 Assistant Professor

2 should be simplistic. Furthermore, the biochemical kit should consists of only AB products at the lowest cost possible. All experiments were done on a StepOnePlus Real-Time PCR System from AB as a benchmark for reactions prior to testing on the prototype. Chemicals tested Enzyme type and concentration is optimized in this paper. Cosolvents those that were shown to improve enzyme efficiency are tested. These are BSA, Tween 2 and dc 7 GTP. DNA polymerase DNA polymerase from Applied biosystems: AmpliTaq, AmpliTaq LD (low DNA) and SuperTaq are used in this experiment. Cosolvents Cosolvents which stabilizes enzyme and facilitates amplification are tested. These are Bovine serum albumin (BSA), Tween 2 and 7-deaza-2'-deoxyguanosine (dc 7 GTP). MATERIALS & METHODS DNA template Two DNA templates of GFP were used in the experiment. pacgfp1-n1 was used for enzyme testing and pemgpf-bsd was used for cosolvent testing. QIAprep Spin Miniprep Kit was used for purification of plasmid DNA from E. coli. Concentration of DNA was estimated using its absorbance at 26nm. PCR reactions Enzyme type The following reagents were used: 1x concentration reaction buffer, 2µM of each dntp,.5µm of forward primer,.5µm of reverse primer,.25u/µl of each DNA polymerase (AmpliTaq, AmpliTaq LD and SuperTaq ), pacgfp1-n1 DNA template of different concentrations (.ng/µl,.6ng/µl. 1.2ng/µl and 2.4ng/µl) and 3µl of SYBR Green. Reactions were done with varying running conditions for each enzyme. Cosolvent DNA concentration of 3ng/µl was used and positive control does not contain any cosolvents. Primer concentration are 1µM each and SuperTaq DNA polymerase at concentration.5u/µl. BSA of concentrations1µg/ml, 5µg/ml, and 1µg/ml; Tween 2 at concentrations.1%,.5% and 1.% and dc 7 GTP of concentrations.2mm,.6mm and 1.mM were tested.

3 RESULTS AND DISCUSSION Enzyme type and concentration SuperTaq is the most efficient as it shows amplification of DNA at a lower C T compared to AmpliTaq LD and SuperTaq. Only enzyme at concentration.5u/µl and.75u/µl shows amplification. Although amplification of higher enzyme concentration at.75u/µl shows more efficient amplification (lower C T value by 5 cycles) and more products (higher ΔRn values) when compared to enzyme concentration at.5u/µl, enzyme of concentrations.5u/µl is used. This is due to the benefit of cost versus efficiency. An increase of enzyme concentration of.5u/µl to.75u/µl would increase cost by 4% to reduce time taken by 3 minutes. Cosolvents A B C -.2 differenc -.4 e in C T value -.6 BSA concentration (µg/ml) against average difference in C T value BSA concentration (µg/ml) Concentration of Tween 2 (%) against average difference in C T value difference in C T Tween 2 concentration (%) C T difference dc 7 GTP concentration (mm) against average C T difference dc 7 GTP concentration (mm) Figure 1. difference in C T values between reactions without cosolvents and different cosolvent concentrations for A)BSA, B)Tween 2 and C) dc 7 GTP BSA Increase in BSA concentrations does not show a significant increase in C T value (Figure 1A).

4 Tween 2 Based on Figure 1B, it can be concluded that the addition of.1% Tween 2 would increase efficiency of PCR reaction. Although the difference in C T values is significant, increase in C T is minimal and only reduces reaction time by 2 seconds. dc7gtp Although the dc 7 GTP is recommended to be used in place of deoxyguanosine trhiphosphate (dgtp) in a ratio of 3:1 (Innis et al, 199), this was not done because deoxynucleotide triphospates (dntps) provided with Supertaq Polymerase is in a mix of 1mM (Applied Biosystems, 29b). An increase in dc 7 GTP concentration reduces amplification indicated by the increasing C T value (Figure 1C). Future experiments It appears that addition of the cosolvents tested does not improve amplification efficiency significantly with the exception of the slight improvement when.1% of Tween 2 was added. However, only single cosolvents have been tested. A mixture of cosolvents may have complementary effects on the PCR reaction and could be tested. Moreover, further experiments have to be done to meet the objective of a robust biochemical kit. Although decreasing cycle number would prevent the significant amplification of contamination, other factors that affect the reaction should be taken into account. For instance, the stability of the reagents used. Almost all reagents need to be kept at -2 C and its properties could be affected when left at room temperature for extended periods. Since the biochemical kit is for educational purposes, the stability of the reagents could be compromised due to lack of proper equipment or carelessness of students. Thus, an experiment to test the effect on the amplification reaction when reagents are left in room temperature for different periods of time can be done. Furthermore, the protocol have to take into account the limitations of the low-cost prototype. At the moment, it takes the prototype 45 seconds for heating from 6 C to 9 C and 1 minute to cool from 9 C to 6 C. This is about 9 to 1 times slower than that of the StepOnePlus Real-Time PCR System used in this experiment. The difference in rate of cooling and heating could affect the amplification efficiency. Therefore, the rate of heating and cooling should be altered in the StepOnePlus Real-Time PCR System to match those of the low-cost prototype. REFERENCES Applied Biosystems (29b). Product Description, SuperTaq and SuperTaq Plus. Extracted from &tab=detailinfo Innis, M. A., Gelfand, D. H., Sninsky, J. J. and White, T. J., eds. (199) PCR Protocols, A Guide to Methods and Application, Academic Press, San Diego, CA.

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