Nucleic Acids Research

The fluorescence polarization has been applied to the monitoring hybridization of a fluorescently labeled probe without separation (1-3). It has also the potential of analysis of PCR process with fluorescently labeled primer. A fluorescent dye tethered to primer will experience slower tumbling (longer correlation time) upon conversion of the primer to PCR product (4). So a PCR process with a dye-modified primer can be monitored without electrophoresis and separation, provided an appreciable percentage of the total primer is converted to PCR product form by Taq polymerase. In this paper, the method based on combination of PCR and fluorescence polarization is described, and PCR product is quantitatively monitored by anisotropy ratio and the mean hydrodynamic volume.

Anisotropy ratio was measured using FP777 spectrofluorimeter equipped with a microcomputer-assisted polarization measurement module and a Peltier temperature regulation system (Jasco, Tokyo). Excitation at 485 nm was provided by passing the output of a 450 W xenon arc lamp through a monochromator with the entrance and exit slits set to a bandpass of 5 nm.

The fluorescence anisotropy is expected to decrease with increasing temperature due to faster Brownian motion, decreased viscosity and decreased excited state lifetime. Anisotropy and the hydrodynamic volume (V) of a fluorescent molecule or molecule assembly in a homogeneous solution are related by the Perrin equation (5) 1/r = 1/r_o (1 + [tau]RT/[eta]V), where r_o is the limiting anisotropy of the molecule, i.e., the anisotropy in the absence of rotation, [tau] is the observed fluorescence lifetime, R is the gas constant, T is absolute temperature and [eta] is the viscosity of the solution at T.

If the fluorescent molecules with different hydrodynamic volumes coexist in a homogeneous solution, r, r_o and V in the Perrin equation will be replaced by the observed anisotropy, the mean limiting anisotropy of the molecules and the mean effective hydrodynamic volume of all fluorescent molecules in the solution, respectively. The estimation was carried out using the linear Perrin plots (1/r versus T/[eta]) of the mean relative hydrodynamic volume (V_rel). The relative volume, which represents an overall comparison of the effective volumes of fluorescently labeled oligodeoxynucleotides in the experiment, is used instead of absolute volume (V_abs), since it is difficult and unnecessary to calculate the absolute volume.

A series of samples to simulate PCR process were prepared using the mixture solution of 10^-8 M of the fluorescently labeled dsDNA fragment and fluorescently labeled primer 1 with the different percentage of the fluorescently labeled dsDNA fragment (as PCR product of stx2 gene 188-443 bp). The fluorescently labeled dsDNA fragment was prepared through asymmetric PCR amplification reaction, which was performed with 20 × 100 µl volumes containing 0.5 mM of each dNTP, 0.01 µM fluorescently labeled primer 1 (5[prime] F-TAAACCACACCCCACCGGGCA-3[prime]), 0.2 µM primer 2 (5[prime]-GAACGATCCAGCGCTGCGACA),genomic DNA of Escherichia coli O157:H7 and 2.5 U Ex Taq (TaKaRa, Japan). The PCR reaction was carried out in the Perkin Elmer GeneAmp PCR System 9600 (Norwalk, CT, USA) for 45 cycles of 30 s at 94°C, 30 s at 63°C and 30 s at 72°C with a final extension at 72°C for 10 min. In this asymmetric PCR, a large fraction of 0.01 µM fluorescently labeled primer 1 was converted into fluorescently labeled dsDNA fragment. The fluorescently labeled dsDNA fragment was further purified by removing the remaining primers.

Anisotropy of the series of samples prepared was measured at 25°C. Results are shown in Figure 1. Anisotropy increases with an increasing percentage of the fluorescently labeled dsDNA fragment in the samples, as expected. The mean relative volumes of the series of samples were calculated through anisotropy temperature effect experiment (5-40°C). Relative volumes are normalized to a volume of unity for the fluorescently labeled primer 1 (data not shown).

Figure 1. The anisotropy of the series of samples to simulate PCR process.

The curve shown in Figure 1 and the plot of the relative volume versus the percentage of fluorescently labeled dsDNA fragment can be served as calibration curves to analyze the conversion ratio of the fluorescently labeled primer 1 to amplification product during PCR.

Next, we applied this method to an actual PCR reaction. The stx2 gene (0.5 mM of each dNTP, 0.01 µM fluorescently labeled primer 1, 0.2 µM primer 2, genomic DNA of E.coli O157:H7 and 2.5 U Ex Taq in a total of 5 × 100 µl solution) was amplified. Thermal cycling was performed with an initial denaturation at 95°C for 3 min, followed by 20-35 cycles at 94°C for 30 s, 63°C for 30 s and 72°C for 30 s. Following this a final extension was carried out at 72°C for 10 min. PCR products were electrophoresed through a 2% agarose gel (as shown in Fig. 2). The anisotropy of PCR product solutions with 20, 25, 30 and 35 cycles was measured at 25°C. The conversion percentage of primer 1 to PCR product was calculated. The results are shown in Table 1.

Figure 2. Electrophoresis of PCR products (3 µl each) was performed on a 2% agarose gel. Lanes 1 and7, DNA size Marker (100, 200, 300, 400, 500, 525, 700 and 1000 bp). Lane 2, E.coli JM109 DNA as template. Lanes 3, 4, 5 and 6, E.coli O157 DNA as template, PCR with 20, 25, 30 and 35 cycles, respectively.

Table 1. The anisotropy ratio and estimated percentage B of stx2 gene PCR product at different number of cycles

Number of cycles	Anisotropy ratio	Percentage (B)
20	0.026	8
25	0.033	15
30	0.058	33
35	0.096	85

The PCR product with 35 cycles was also studied using the anisotropy temperature effect, and the relative volume was estimated through the Perrin plot of 1/r versus T/[eta]. The experimental results suggested that the primer 1 of ~89% had been converted into the PCR product (~9 nM) after PCR reaction of 35 cycles.

Through the analysis using two parameters, it was observed that for the PCR of stx2 gene (188-443 bp fragment) with 0.01 µM primer 1, 0.2 µM primer 2 and 35 cycles, the primer 1 of 85-89% was converted to PCR product. The results are consistent with the values obtained by ethidium bromide staining of gel and scanning densitometry (data not shown).

We have combined the PCR technique with fluorescence polarization using a fluorescently labeled primer. The anisotropy and the mean relative volume of the fluorescently labeled DNA (F-primer and F-dsDNA fragment) in PCR product solution were determined. The conversion percentage of fluorescently labeled primer to the PCR product is monitored through an increase in anisotropy and the mean relative volume. The present work demonstrated the potential of the technique in quantitative analysis of PCR process. The commercial FA equipment can directly be used in anisotropy analysis method. For mean relative volume analysis, FA equipment with temperature regulation is necessary, i.e., Beacon 2000 FA system.

ACKNOWLEDGEMENT

The authors are grateful to the Department of Bacterial Infections Research Institute for Microbial Disease (Osaka University) for providing the genome DNA of E.coli O157:H7.

REFERENCES

1. Kumke,M.U., Li,G., McGown,L.B., Walker,G.T. and Linn,C.P. (1995) Anal. Chem., 67, 3945-3951. MEDLINE Abstract

2. Murakami,A., Nakaura,M., Nakatsuji,S., Tran-Cong,Q. and Makino,K. (1991) Nucleic Acids Res., 19, 4097-4102. MEDLINE Abstract

3. Kumke,M.U., Shu,L., McGown,L.B., Walker,G.T., Pitner,J.B. and Linn,C.P. (1997) Anal. Chem., 500-506. MEDLINE Abstract

4. Walker,G.T., Linn,C.P. and Nadeau,J.G. (1996) Nucleic Acids Res., 24, 348-353. MEDLINE Abstract

5. Cantor,C.R. and Schimmel,P.R. (1980) Biophysical Chemistry Part II: Techniques for the Study of Biological Structure and Function.W.H. Freeman & Co., San Francisco, CA.

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*To whom correspondence should be addressed. Tel: +86 21 64253823; Fax: +86 21 64253904; Email: jjzhong@ecust.edu.cn

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