Bio/Technology
11, 1026 - 1030 (1993)
doi:10.1038/nbt0993-1026
Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification ReactionsRussell Higuchi1, *, Carita Fockler1, Gavin Dollinger2
& Robert Watson1
1Roche Molecular Systems, Inc., 1145 Atlantic Ave., Alameda, CA 94501.
2Chiron Corporation, 1400 53rd St., Emeryville, CA 94608.
*Corresponding author. We describe a simple, quantitative assay for any amplifiable DNA sequence that uses a video camera to monitor multiple polymerase chain reactions (PCRs) simultaneously over the course of thermocycling. The video camera detects the accumulation of double-stranded DNA (dsDNA) in each PCR using the increase in the fluorescence of ethidium bromide (EtBr) that results from its binding duplex DNA. The kinetics of fluorescence accumulation during thermocycling are directly related to the starting number of DNA copies. The fewer cycles necessary to produce a detectable fluorescence, the greater the number of target sequences. Results obtained with this approach indicate that a kinetic approach to PCR analysis can quantitate DNA sensitively, selectively and over a large dynamic range. This approach also provides a means of determining the effect of different reaction conditions on the efficacy of the amplification and so can provide insight into fundamental PCR processes.
REFERENCES
- Mullis, K., Faloona, R., Scharf, S., Saiki, R., Horn, G. and Erlich, H. 1986. Specific enzymatic amplification of DNA in vitro: The polymerase chain reaction. Cold Spring Harb. Symp. Quant. Biol. 51: 263−273. | ChemPort |
- Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. 1988. Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase. Science 239: 487−491. | PubMed | ISI | ChemPort |
- Higuchi, R., Dollinger, G., Walsh, P.S. and Griffith, R. 1992. Simultaneous amplification and detection of specific DNA sequences. Bio/Technology 10: 413−417. | PubMed | ISI | ChemPort |
- Sutherland, J.C., Sutherland, B.M., Emrick, A., Monteleone, D.C., Ribeiro, E.A., Trunk, J., Son, M., Serwer, P., Poddar, S.K. and Maniloff, J. 1991. Quantitative electronic imaging of gel fluorescence with CCD cameras: Applications in molecular biology. Biotechniques 10: 492−497. | PubMed | ISI | ChemPort |
- Kwok, S.Y., Mack, D.H., Mullis, K.B., Poiesz, B.J., Ehrlich, G.D., Blair, D. and Friedman-Kien, A.S. 1987. Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection. J. Virol. 61: 1690−1694. | PubMed | ISI | ChemPort |
- Lawyer, F.C., Stoffel, S., Saiki, R.K., Chang, S., Landre, P., Abramson, R. and Gelfand, D.H. 1993. High-level expression, purification, and enzymatic characterization of full-length Thermits aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity. PCR Journal. In press.
- Walsh, P.S., Metzger, D.A. and Higuchi, R. 1991. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 10: 506−513. | PubMed | ISI | ChemPort |
- Higuchi, R. and Kwok, S. 1989. Avoiding false positives with PCR. Nature 339: 237−238. | Article | PubMed | ISI | ChemPort |
- Piatak, M. Jr., Saag, M.S., Yang, L.C., Clark, S.J., Kappes, J.C., Luk, K.-C., Hahn, B.H., Shaw, G.M. and Lifson, J.D. 1993. High levels of HIV-1 plasma during all stages of infection determined by competitive PCR. Science 259: 1749−1754. | PubMed | ISI | ChemPort |
- McCarrey, J.R., Dilworth, D.D. and Sharp, R.M. 1992. Semiquantitative analysis of X-linked gene expression during spermatogenesis in the mouse: Ethidium-bromide staining of RT-PCR products. Genet. Anal. Tech. Appl. 9: 117−123. | Article | PubMed | ChemPort |
- Gilliland, G., Perrin, S., Blanchard, K. and Bunn, H.F. 1990. Analysis of cytokine mRNA and DNA: Detection and quantitation by competitive polymerase chain reaction. Proc. Natl. Acad. Sci. 87: 2725−2729. | PubMed | ChemPort |
- Rye, H.S., Yue, S., Wemmer, D.E., Quesada, M.A., Haugland, R.P., Mathies, R.A. and Glazer, A.N. 1992. Stable florescent complexes of double-stranded DNA with bis-intercalating asymmetric czanimol dyes: properties and applications. Nucl. Acids Res. 20: 2803−2812. | PubMed | ISI | ChemPort |
- Holland, P.M., Abramson, R.D., Watson, R. and Gelfand, D.H. 1991. Detection of specific polymerase chain reaction product by utilizing the 5'
 3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. 88: 7276−7280. | PubMed | ChemPort |
- Chou, Q., Russell, M., Birch, D.E., Raymond, J. and Bloch, W. 1992. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number simplifications. Nucl. Acids Res. 20: 1717−1723. | PubMed | ISI | ChemPort |
- Kwok, S., Kellogg, D.E., McKinney, N., Spasic, D., Goda, L., Levenson, C. and Sninsky, J.J. 1990. Effects of primer-template mismatches on the polymerase chain reaction: Human immunodeficiency virus type 1 model studies. Nucl. Acids Res. 18: 999−1005. | PubMed | ISI | ChemPort |
- Jackson, J.B., Ndugwa, C., Mmiro, F., Kataaha, P., Guay, L., Dragon, E.A., Goldfarb, J. and Olness, K. 1991. Non-isotopic polymerase chain reaction methods for the detection of HIV-1 in Ugandan mothers and infants. AIDS 5: 1463−1467. | PubMed | ISI | ChemPort |
- Sanchez-Pescador, R., Power, M.D., Barr, P.J., Steimer, K.S., Stempien, M.M., Brown-Shimer, S.L., Gee, W.W., Renard, A., Randolph, A., Levy, J.A., Dina, D. and Luciw, P.A. 1985. Nucleotide sequence and expression of an AlDS-associated retrovirus (ARV-2). Science 227: 484−492. | PubMed | ChemPort |
- Horn, G.T., Bugawan, T.L., Long, C.M. and Erlich, H.A. 1988. Allelic sequence variation of the HLA-DQ
 loci, relationship to serology and to insulin-dependent diabetes susceptibility. Proc. Natl. Acad. Sci. 85: 3504−3508. | PubMed |
|
