Eppendorf BioNews Application Notes
A better way to optimize: Two-step Gradient PCR Joel Lopez and Vincent Prezioso, BioSystems Laboratory, Brinkmann™ Instruments, Inc., Westbury, NY, USA Introduction Science underwent revolutionary changes during the 1980’s, particularly in the field of genetics. One of the most significant changes was inspired by the 1985 article by Saiki, et al., in which the amplification of specific beta-globin sequences and the subsequent restriction analysis for diagnosis of sickle cell anemia is described [1]. The technique referenced utilized biological and chemical components to orchestrate an enzymatic amplification reaction conducted by a DNA polymerase. This technique was named the Polymerase Chain Reaction (PCR). This article describes an important extension of the PCR technique: Two-step Gradient PCR. This method offers significant time-savings and minimizes reagent use, relative to a standard PCR optimization protocol. This method is applied to the standardizing of the amplification of a segment of mouse tissue plasminogen activator (tPA) cDNA, using an oligonucleotide set. The calculated melting temperatures for this amplification were 57.3 °C for K2-RC, and 57.8 °C for GF-ATG [2 ]. The standardization of the reactions was performed using the Eppendorf ® Mastercycler ® gradient [3 ], which provided excellent, reproducible, and rapid results.
Time
94 °C
2 min
0.5 mM MgCl2
94 °C
15 sec
0.1 mM dNTP
50 - 70.5 °C
30 sec
10 pmol per primer
72 °C
1 min
1.25 units Taq Polymerase [ 5]
4 °C
Hold
Oligonucleotide* primers used for the amplification of the mouse tissue plasminogen activator cDNA fragment: 5’-AGGTGGACTCGAGGCATGGGGAC-3’ K2-RC 5’-GTCCGAAGTCATATGTGCAGCGAACCAAG-3’ GF-ATG
*Oligonucleotides were obtained as custom synthesis products from Integrated DNA Technologies, Inc. [6 ]
Each PCR reaction was performed in a single PCR tube. Reactions were carried out using the Eppendorf Mastercycler gradient and Eppendorf Taq Polymerase. 9 µl of each reaction was resolved in a 1% agarose gel for a period of 1.5 h at 80 V. 10X DNA Gel Loading Buffer [7] was added at a ratio of 9:1.
2) Two-step Gradient PCR, 40 °C - 60.6 °C
Results and Discussion
Next, a two-step gradient experiment with a temperature range of 40 °C to 60.6 °C was performed to confirm the temperature optima obtained in Fig. 1, and to determine the lower limit of the annealing /elongation temperature. Using this gradient, an
1) Two-step Gradient PCR, 50 °C - 70.5 °C In the first experiment, a two-step gradient PCR experiment was performed at temperatures between 50 °C and 70.5 °C. Fig. 1 shows clearly that the temperatures 1
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This method incorporates a two-step protocol (combining the annealing and elongation steps), which is designed for significant time-savings and a reduction in reagent use during optimization and standard PCR experiments.
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1,000 bp 850 bp 650 bp
Fig. 1b
30x
between 50 °C and 60.8 °C work well for this PCR (lanes 2 to 8, Fig . 1a). Plotting the relative intensities from the analysis of the PCR products, it is evident that the higher intensities are in the range of 50 °C and 60.8 °C (Fig. 1b). At temperatures higher than 60.8 °C, a pattern of non-specific product amplification appeared, where the intensity of that product was inversely proportional with the intensity of the specific product (Fig. 1b). While a conclusive reason for this non-specific amplification product could not be determined, it was extremely reproducible at those temperatures under a variety of reaction conditions (not shown).
2,000 bp 1,650 bp
The presentation of a practical method that reduces the time devoted to uncovering optimal annealing temperatures.
Mouse tPA cDNA cloned in pBluescript KS [4] was used with oligonucleotides designed approximately 650 bp apart. The reaction conditions were as follows:
Two-step PCR Program
Fig. 1a
Objective
Materials and methods
Reaction mix (50 µl) 0.05 ng of Template
1% Agarose gel stained with ethidium bromide. Lane 1, 1 kb DNA ladder [8]. Lanes 2 to 13, Two-step Gradient PCR, temperatures from 50 °C to 70.5 °C. Lane 2, 50 °C; 3, 50.3 °C; 4, 51.4 °C; 5, 53.2 °C; 6, 55.5 °C; 7, 58.1°C; 8, 60.8 °C; 9, 63.5 °C; 10, 66 °C; 11, 68.1 °C; 12, 69.7 °C; 13, 70.5 °C Specific Product Non-specific Product
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Histogram of the analysis of the relative intensity of PCR products, specific and non-specific bands using Kodak 1D Image Analysis software [ 9].
60 Relative Intensity 40 20
Specific Product Non-specific Product
0 50
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55.5 60.8 Temperature °C
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Eppendorf BioNews Application Notes
A better way to optimize: Two-step Gradient PCR
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Fig. 2a
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2,000 bp 1,650 bp
1% Agarose gel stained with ethidium bromide. Lane 1, 1 kb DNA ladder [8 ]. Lanes 2 to 13, Gradient Two-step PCR, temperatures from 40 °C to 60.6 °C. Lane 2, 40 °C; 3, 40.2 °C; 4, 41.3 °C; 5, 43.1 °C; 6, 45.4 °C; 7, 48 °C; 8, 50.7 °C; 9, 53.5 °C; 10, 56 °C; 11, 58.1 °C; 12, 59.8 °C; 13, 60.6 °C.
1,000 bp 850 bp 650 bp
Fig. 2b
Specific Product Non-specific Product
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Histogram of the analysis of the relative intensity of PCR products, specific and non-specific bands using Kodak 1D Image Analysis software [9].
40 Relative Intensity 20
Specific Product Non-specific Product
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45.4 50.7 Temperature °C
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excellent amplification was achieved in the range of 48°C to 60.6°C (Fig. 2). The range of 48 °C to 60.6 °C provided superior temperatures due to the robust, unique, and clean products obtained during the amplification, without any secondary products such as primer-dimers. Robust products were obtained at temperatures lower than 48 °C, but also evidenced were artifacts at molecular weights slightly lower than the expected product (Fig. 2a). The artifacts showed higher intensity at lower temperatures. These artifacts may have resulted from mis-priming at lower temperatures.
obtained at temperatures of 50.3 °C and 50.7 °C. A final experiment was performed to confirm that these temperatures were optimal for the amplification of this fragment of the Mouse tPA gene (Fig. 3). A two-step PCR was performed with the annealing/elongation temperature set to 50.5 °C. This temperature resulted in excellent amplification, as evidenced by the robust, unique, and clean products obtained, without any secondary products such as primer-dimers.
This experiment showed that temperatures between 50 °C and 60.8 °C (Fig. 1) provide a viable range for annealing / extension steps, resulting in unique and
Intensity plots from both sets of experiments reveal the best product yield was 1
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1% Agarose gel stained with ethidium bromide. Lane 1, 1 kb DNA ladder [ 8 ]. Lanes 2 to 13, Two-step PCR. Temperature at 50.5 °C.
2,000 bp 1,650 bp 1,000 bp 850 bp 650 bp
Overall, the best temperatures for obtaining products of outstanding quality were in the range of 50.7 °C to 60.6 °C (Fig. 2). The intensity plots from both sets of experiments reveal that the best yield of product was acquired at temperatures of 50.3 °C and 50.7 °C, respectively. This was confirmed by performing a two-step PCR at a temperature of 50.5 °C (Fig. 3).The small difference in the annealing/extension temperature corroborates the accuracy of the PCR reactions using the Eppendorf Mastercycler gradient. It was noted that the most intense products were not necessarily of the highest quality, as they could be contaminated with artifacts. The results of this experiment also show that two-step PCR can be performed at lower temperatures than are normally recommended for this technique. Usually, two-step experiments are not attempted unless the annealing temperatures of the primers are 65 °C or higher. This experiment has verified that a two-step PCR can be performed at temperatures as low as 50.3 °C. Further experimentation may lower this threshold further, showing that most PCR reactions can be performed in two steps, saving researchers considerable time. Acknowledgments: The authors would like to thank Dr. Stella Tsirka of SUNY at Stony Brook for the tPA cDNA clone, and helpful discussions. References
Conclusion
3) Two-step PCR, 50.5 °C
Fig. 3
59.8
clean products. Similar results were obtained for the gradient of 40 °C to 60.6 °C.
Specific Product
[1] Saiki, R.K.; Scharf, S.J.; Faloona, F.; Mullis, K.B.; Horn, G.T.; Erlich, H.A.; and Arnheim, N. 1985. “Enzymatic amplification of beta-globin sequences and restriction site analysis for diagnosis of sickle cell anemia.” Science, 230: 1350-1354. [2] Sharroks, A.D. 1994. “The design of primers for PCR,” PCR Technology, Current Innovations, Griffin, H.G., and Griffin, A.M., Ed., CRC Press, London, 5-11. [3] Mastercycler gradient, Eppendorf AG. [4] Rickles, R.J.; Darrow, A.L.; and Strickland, S. 1988. “Molecular cloning complementary DNA to mouse tissue plasminogen activator mRNA and its expression during F9 teratocarcinoma cell differentiation.” J. Biol. Chem., Vol. 263 (3): 1563-1569. [5] Taq DNA Polymerase*, Eppendorf AG. [6] Integrated DNA Technologies, Inc., Coralville, IA, USA. [7] 10x DNA Gel Loading Buffer, Eppendorf AG. [8] 1 kb Plus DNA Ladder, Invitrogen, Carlsbad, CA, USA. [9] Kodak 1D Image Analysis Software, 2000. * Eppendorf Taq polymerase is sold under licensing arrangements with F. Hoffmann-La Roche Ltd, Roche Molecular Systems, Inc. and The Perkin-Elmer Corporation.
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