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Two-step Gradient PCR*

Joel Lopez and Vincent Prezioso, PhD
BioSystems Laboratory, Brinkmann Instruments Inc., Westbury, New York 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 techniqueTwo-step Gradient PCR. This method offers significant time-savings and minimizes reagent use, relative to a standard PCR optimization protocol. The 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 57.3C for K2-RC, and 57.8C for GF-ATG (2). The standardization of the reactions was performed using the Eppendorf Mastercycler gradient (3), which provided excellent, reproducible, and rapid results.

Objective

The presentation of a practical method that reduces the time devoted to uncovering optimal annealing temperatures. This method incorporates a two-step protocol (combining the annealing and elongation steps), which is designed for significant time-savings and a reducti on in reagent use during optimization and standard PCR experiments.

Materials and Methods

Mouse tPA cDNA cloned in pBluescript KS (4) was used with oligonucleotides designed approximately 650 bp apart. The reaction conditions were as follows:

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. Nine micro liters of each reaction was resolved in a 1% agarose gel for a period of one and a half hours at 80 volts. 10x DNA Gel Loading Buffer (7) was added at a ratio of 9:1. Results and Discussion

Two-step Gradient PCR, 50C to 70.5C.

Figure 1A. 1% Agarose gel stained with ethidium bromide.
Lane 1, 1 kb DNA ladder (8).
Lanes 2 to 13, Two-step Gradient PCR, temperatures from 50C to 70.5C.
Lane Figure 1B. Histogram of the analysis of the relative intensity of PCR products, specific and non-specific bands using Kodak 1D Image Analysis software (9) 2, 50C
3, 50.3C
4, 51.4C
5, 53.2C
6, 55.5C
7, 58.1C
8, 60.8C
9, 63.5C
10, 66.0C
11, 68.1C
12, 69.7C
13, 70.5C
In the first experiment, a two-step gradient PCR experiment was performed at temperatures between 50C and 70.5C. Figure 1 shows clearly that the temperatures between 50C and 60.8C work well for this PCR (lanes 2 to 8, Figure 1a). Plotting the relative intensities from the analysis of the PCR products, it is evident that the higher intensities are in the range of 50C and 60.8C (Figure 1b). At temperatures higher than 60.8C, a pattern of non-specific product amplification appeared, where the intensity of that product was inversely proportional with the intensity of the specific product (Figure 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).

Two-step Gradient PCR, 40C to 60.6C.

Figure 2A. 1% Agarose gel stained with ethidium bromide.
Lane 1, 1 kb DNA ladder (8).
Lanes 2 to 13, Gradient Two-step PCR, temperatures from 40C to 60.6C. .
Lane

Figure 2B. Histogram of the analysis of the relative intensity of PCR products, specific and non-specific bands using Kodak 1D Image Analysis software (9) 2, 40C
3, 40.2C
4, 41.3C
5 , 43.1C
6, 45.4C
7, 48.0C
8, 50.7C
9. 53.5C
10, 56.0C
11, 58.1C
12, 59. 8C
13, 60.6C
Next, a two-step gradient experiment with a temperature range of 40.0C to 60.6C was performed to confirm the temperature optima obtained in Figure 1, and to determine the lower limit of the annealing/elongation temperature. Using this gradient, an excellent amplification was achieved in the range of 48.0C to 60.6C (Figure 2). The range of 48.0C to 60.6C 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 48.0C, but also evidenced were artifacts at molecular weights slightly lower than the expected product (Figure 2a). The artifacts showed higher intensity at lower temperatures. These artifacts may have resulted from mis-priming at lower temperatures.

Two-step PCR, 50.5C

Figure 3. 1% Agarose gel stained with ethidium bromide.
Lane 1, 1 kb DNA ladder (8).
Lanes 2 to 13, Two-step PCR. Temperature at 50.5C.

Intensity plots from both sets of experiments reveal the best product yield was obtained at temperatures of 50.3C and 50.7C. A final experiment was performed to confirm that these temperatures were optimal for the amplification of this fragment of the Mouse tPA gene (Figure 3). A two-step PCR was performed w ith the annealing/elongation temperature set to 50.5C. This temperature resulted in excellent amplification, as evidenced by the robust, unique, and clean products obtained, without any secondary products such as primer-dimers. Conclusion

This experiment evidenced that temperatures between 50C and 60.8C (Figure 1) provide a viable range for annealing/extension steps, resulting in robust, unique, and clean products. Similar results were obtained for the gradient of 40C to 60.6C.

Overall, the best temperatures for obtaining products of outstanding quality were in the range of 50.7C to 60.6C (Figure 2). The intensity plots from both sets of experiments reveal that the best yield of product was acquired at temperatures of 50.3C and 50.7C, respectively. This was confirmed by performing a two-step PCR at a temperature of 50.5C (Figure 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 65C or higher.

This experiment has verified that a two-step PCR can be performed at temperatures as low as 50.3C. 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.

Trademark and Disclaimer Information
Brinkmann is a trademark of Brinkmann Instruments, Inc.
Eppendorf and Mastercycler are registered trademarks of Eppendorf AG.
*PCR Disclaimer: PCR is licensed under U.S. patent numbers 4,683,202, 4,683,195, 4,965,188 and 5,075,216 or their foreign counterparts, owned by Hoffmann-La Roche Inc. and F. Hoffmann-La Roche Ltd.

References
  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.
  7. 10x DNA Gel Loading Buffer, Eppendorf AG.
  8. 1 kb Plus DNA Ladder, Life Technologies.
  9. Kodak 1D Image Analysis Software, 2000.

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