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. 1997 May 15;25(10):1999–2004. doi: 10.1093/nar/25.10.1999

Nanoliter scale PCR with TaqMan detection.

O Kalinina 1, I Lebedeva 1, J Brown 1, J Silver 1
PMCID: PMC146692  PMID: 9115368

Abstract

We monitored PCR in volumes of the order of 10 nl in glass microcapillaries using a fluorescence energy transfer assay in which fluorescence increases if product is made due to template-dependent nucleolytic degradation of an internally quenched probe (TaqMan assay). This assay detected single starting template molecules in dilutions of genomic DNA. The results suggest that it may be feasible to determine the number of template molecules in a sample by counting the number of positive PCRs in a set of replicate reactions using terminally diluted sample. Since the assay system is closed and potentially automatable, it has promise for clinical applications.

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Selected References

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  1. Becker-André M., Hahlbrock K. Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acids Res. 1989 Nov 25;17(22):9437–9446. doi: 10.1093/nar/17.22.9437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burns M. A., Mastrangelo C. H., Sammarco T. S., Man F. P., Webster J. R., Johnsons B. N., Foerster B., Jones D., Fields Y., Kaiser A. R. Microfabricated structures for integrated DNA analysis. Proc Natl Acad Sci U S A. 1996 May 28;93(11):5556–5561. doi: 10.1073/pnas.93.11.5556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cheng J., Shoffner M. A., Hvichia G. E., Kricka L. J., Wilding P. Chip PCR. II. Investigation of different PCR amplification systems in microbabricated silicon-glass chips. Nucleic Acids Res. 1996 Jan 15;24(2):380–385. doi: 10.1093/nar/24.2.380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gerard C. J., Arboleda M. J., Solar G., Mulé J. J., Kerr W. G. A rapid and quantitative assay to estimate gene transfer into retrovirally transduced hematopoietic stem/progenitor cells using a 96-well format PCR and fluorescent detection system universal for MMLV-based proviruses. Hum Gene Ther. 1996 Feb 10;7(3):343–354. doi: 10.1089/hum.1996.7.3-343. [DOI] [PubMed] [Google Scholar]
  5. Gibson U. E., Heid C. A., Williams P. M. A novel method for real time quantitative RT-PCR. Genome Res. 1996 Oct;6(10):995–1001. doi: 10.1101/gr.6.10.995. [DOI] [PubMed] [Google Scholar]
  6. Gilliland G., Perrin S., Blanchard K., Bunn H. F. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2725–2729. doi: 10.1073/pnas.87.7.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Heid C. A., Stevens J., Livak K. J., Williams P. M. Real time quantitative PCR. Genome Res. 1996 Oct;6(10):986–994. doi: 10.1101/gr.6.10.986. [DOI] [PubMed] [Google Scholar]
  8. Higuchi R., Dollinger G., Walsh P. S., Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (N Y) 1992 Apr;10(4):413–417. doi: 10.1038/nbt0492-413. [DOI] [PubMed] [Google Scholar]
  9. Holland P. M., Abramson R. D., Watson R., Gelfand D. H. Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7276–7280. doi: 10.1073/pnas.88.16.7276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Livak K. J., Flood S. J., Marmaro J., Giusti W., Deetz K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 1995 Jun;4(6):357–362. doi: 10.1101/gr.4.6.357. [DOI] [PubMed] [Google Scholar]
  11. Sninsky J. J., Kwok S. The application of quantitative polymerase chain reaction to therapeutic monitoring. AIDS. 1993 Nov;7 (Suppl 2):S29–S34. doi: 10.1097/00002030-199311002-00007. [DOI] [PubMed] [Google Scholar]
  12. Wittwer C. T., Garling D. J. Rapid cycle DNA amplification: time and temperature optimization. Biotechniques. 1991 Jan;10(1):76–83. [PubMed] [Google Scholar]
  13. Wittwer C. T., Ririe K. M., Andrew R. V., David D. A., Gundry R. A., Balis U. J. The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. Biotechniques. 1997 Jan;22(1):176–181. doi: 10.2144/97221pf02. [DOI] [PubMed] [Google Scholar]
  14. Woolley A. T., Hadley D., Landre P., deMello A. J., Mathies R. A., Northrup M. A. Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device. Anal Chem. 1996 Dec 1;68(23):4081–4086. doi: 10.1021/ac960718q. [DOI] [PubMed] [Google Scholar]
  15. Woolley A. T., Mathies R. A. Ultra-high-speed DNA fragment separations using microfabricated capillary array electrophoresis chips. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11348–11352. doi: 10.1073/pnas.91.24.11348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Xu X. H., Yeung E. S. Direct measurement of single-molecule diffusion and photodecomposition in free solution. Science. 1997 Feb 21;275(5303):1106–1109. doi: 10.1126/science.275.5303.1106. [DOI] [PubMed] [Google Scholar]

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