Abstract
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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
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.
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.
Higuchi, R., Dollinger, G., Walsh, P.S. and Griffith, R. 1992. Simultaneous amplification and detection of specific DNA sequences. Bio/Technology 10: 413–417.
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.
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.
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.
Higuchi, R. and Kwok, S. 1989. Avoiding false positives with PCR. Nature 339: 237–238.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Horn, G.T., Bugawan, T.L., Long, C.M. and Erlich, H.A. 1988. Allelic sequence variation of the HLA-DQα loci, relationship to serology and to insulin-dependent diabetes susceptibility. Proc. Natl. Acad. Sci. 85: 3504–3508.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Higuchi, R., Fockler, C., Dollinger, G. et al. Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions. Nat Biotechnol 11, 1026–1030 (1993). https://doi.org/10.1038/nbt0993-1026
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/nbt0993-1026
This article is cited by
-
Smart-Plexer: a breakthrough workflow for hybrid development of multiplex PCR assays
Communications Biology (2023)
-
Assessment of ACC and P450 Genes Expression in Wild Oat (Avena ludoviciana) in Different Tissues Under Herbicide Application
Biochemical Genetics (2023)
-
Prospecting true ScYLV resistance in Saccharum hybrid parental population in India by symptom phenotyping and viral titre quantification
3 Biotech (2023)
-
Identifying optimal reference genes for gene expression studies in Eurasian spruce bark beetle, Ips typographus (Coleoptera: Curculionidae: Scolytinae)
Scientific Reports (2022)
-
Evaluation validation of a qPCR curve analysis method and conventional approaches
BMC Genomics (2021)
