This page contains a Flash digital edition of a book.
Reports


and dsDNA-specific dyes, product purity could be estimated by melting curves. With rapid temperature control, absolute product concentration could be determined by product- to-product annealing kinetics. The only requirements are fluorescence monitoring, the ability to change temperatures rapidly and strict intra-sample temperature homogeneity. Aspects of instrument design are discussed elsewhere (23). Conventional end-point analysis of DNA


amplification by gel electrophoresis identifies product size and estimates purity. However, because amplification is at first stochastic, then exponential, and finally stagnant, the utility of end-point analysis is limited for quantifi- cation. Fluorescence monitoring every cycle during DNA amplification is an extraordi- narily powerful technique for quantification. With simple instrumentation and fluorescent monitoring each cycle, sequence-specific detection and quantification can be achieved in 5–20 min aſter temperature cycling has begun. Although the final fluorescence signal is decreased when low copy numbers are amplified, quantification between 0 and 1000 initial template copies appears possible (Figures 3 and 4). Tese techniques should be particularly useful in assays where rapid quanti- fication is desired, such as in the amplification of clinical serum viruses.


Acknowledgments


Tis work was financially supported by an STTR grant from the NIH (1 R41 GM51647), a Technology Innovation grant from the University of Utah Research Foundation, a Biomedical Engineering grant from the Whitaker Foundation, Idaho Technology, and Associated Regional and University Pathologists. We thank Marla Lay, Gundi Reed, Douglas Searles and Charles Hussey for technical assistance and insightful conversation.


References


1.Barnes, W.M. 1994. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc. Natl. Acad. Sci. USA 91:2216-2220.


2.Gustafson, C.E., R.A. Alm and T.J. Trust. 1993. Effect of heat denaturation of target DNA on the PCR amplification. Gene 123:241-244.


3.Higuchi, R., G. Dollinger, P.S. Walsh and R. Griffith. 1992. Simultaneous amplification and detection of specific DNA sequences. Bio/ Technology 10:413-417.


4.Higuchi, R., C. Fockler, G. Dollinger and R. Watson. 1993. Kinetic PCR analysis: realtime monitoring of DNA amplification reactions. Bio/Technology 11:1026-1030.


5.Holland, P.M., R.D. Abramson, R. Watson and D.H. Gelfand. 1991. Detection of specific


Vol. 54 | No. 6 | 2013


polymerase chain reaction product by utilizing the 5´ to 3´ exonuclease activity of Termus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88:7276-7280.


6.Ishiguro, T., J. Saitch, H. Yawata, H. Yamagishi, S. Iwasaki and Y. Mitoma.1995. Homogeneous quantitative assay of hepatitis C virus RNA by polymerase chain reaction in the presence of a fluorescent intercalater. Anal. Biochem. 229:207-213.


7.Kramer, R.K. and S. Tyagi.1996. Molecular beacons: probes that fluoresce upon hybridization. Nature Biotech. 14:303-308.


8.Lee, L.G., C.R. Connell and W. Bloch.1993. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res. 21:3761-3766.


9.Livak, K.J., S.J.A. Flood, J. Marmaro, W. Giusti and K. Deetz. 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl.4:357-362.


10.Morrison, L.E. 1992. Detection of energy transfer and fluorescence quenching, p. 311- 352. In L.J. Kricka (Ed.), Nonisotopic DNA Probe Techniques. Academic Press, San Diego.


11.Mujumdar, R.B., L.A. Ernst, S.R. Mujumdar and A.S. Waggoner. 1989. Cyanine dye labeling reagents containing isothiocyanate groups. Cytometry 10:11-19.


12.Odelberg, S.J. and R. White. 1993. A method for accurate amplification of polymorphic CA-repeat sequences. PCR Methods Appl. 3:7-12.


13.Roederer, M., A.B. Kantor, D.R. Parks and L.A. Herzenberg. 1996. Cy7PE and Cy7APC: bright new probes for immunofluorescence. Cytometry 24:191-197.


14.Saiki, R.K., D.H. Gelfand, S. Stoffel, S.J. Scharf, R. Higuchi, G.T. Horn, K.B. Mullis and H.A. Erlich.1988. Primer-directed enzymatic ampli- fication of DNA with a thermostable DNA polymerase. Science 239:487- 491.


15.Swerdlow, H., K. Dew-Jager and R.F. Gesteland. 1993. Rapid cycle sequencing in an air thermal cycler. BioTechniques 15:512- 519.


16.Tan, S.T. and J.H. Weis. 1992. Development of a sensitive reverse transcriptase PCR assay, RT-PCR, utilizing rapid cycle times. PCR Methods Appl.2:137-143.


17.Wallace, D.M. 1987. Large- and small-scale phenol extractions and precipitation of nucleic acids, p. 33-48. InS.L. Berger and A.R. Kimmel (Eds.), Guide to Molecular Cloning Techniques (Methods in Enzymology, Vol. 152). Academic Press, Orlando.


18.Wittwer, C.T., G.C. Fillmore and D.J. Garling. 1990. Minimizing the time required for DNA amplification by efficient heat transfer to small samples. Anal. Biochem. 186:328- 331.


19.Wittwer, C.T., G.C. Fillmore and D.R. Hillyard. 1989. Automated polymerase chain reaction in capillary tubes with hot air. Nucleic Acids Res. 17:4353-4357.


20.Wittwer, C.T. and D.J. Garling.1991. Rapid cycle DNA amplification: time and temper- ature optimization. BioTechniques 10:76-83.


21.Wittwer, C.T., B.C. Marshall, G.B. Reed and J.L. Cherry. 1993. Rapid cycle allelespecific amplification: studies with the cystic fibrosis delta F508 locus. Clin. Chem. 39:804- 809.


320 www.BioTechniques.com


22.Wittwer, C.T., G.B. Reed and K.M. Ririe. 1994. Rapid cycle DNA amplification, p. 174- 181. In K.B. Mullis, F. Ferre and R.A. Gibbs (Eds.), Te Polymerase Chain Reaction. Birkhauser, Boston.


23.Wittwer, C.T., K.M. Ririe, R.V. Andrew, D.A. David, R.A. Gundry and U.J. Balis. 1997. Te LightCycler: a microvolume multisample fluorimeter with rapid temperature control. BioTechniques 22:176-181.


24.Wu, P. and L. Brand.1994. Resonance energy transfer: methods and applications. Anal. Biochem. 218:1-13.


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68