Quantitation of Rare DNAs by PCR

Donald M. Coen1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.21
DOI:  10.1002/0471142735.im1021s24
Online Posting Date:  May, 2001
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Abstract

This unit presents a protocol that uses the polymerase chain reaction (PCR) to quantitate the numbers of a particular DNA sequence from 1 to 20,000 molecules per sample. In addition, it helps assess the presence of contaminating sequences, the bane of this kind of procedure. The DNA of interest is prepared and its concentration is determined. A known amount of this DNA is then mixed with two sets of oligonucleotide primers, one set specific for the DNA of interest (e.g., a virus) and the other set specific for an internal control (e.g., a single‚Äźcopy gene encoded by the host organism). The sequences between the primers are amplified, electrophoresed on a gel, transferred to a filter, and probed with oligonucleotides specific for each amplified product. The amounts of the amplified products from the DNA of interest can then be quantitated by comparison to the internal control. For simplicity, the protocol is written in terms of quantitating viral DNA molecules relative to host cellular sequences; however, it can be adapted readily for other applications.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1:

  Materials
  • Cells or tissue sample of interest
  • recipeProteinase digestion buffer (see recipe)
  • 20 mg/ml proteinase K (store at −20°C)
  • Phenol buffered with 50 mM Tris⋅Cl/10 mM EDTA, pH 7.4 (store at room temperature; see unit 10.1 for buffering procedure)
  • 24:1 chloroform/isoamyl alcohol (unit 10.1)
  • 10 M ammonium acetate
  • Cold 100% ethanol
  • 70% ethanol
  • TE buffer, pH 7.5 ( appendix 2A)
  • Internal control DNA from uninfected cells or tissues (see Critical Parameters)
  • DNA molecular weight markers (unit 10.4)
  • recipeReaction mix cocktail (see recipe)
  • Mineral oil
  • 0.8 U/µl Taq DNA polymerase
  • recipeEnd‐labeled oligonucleotide primers for hybridization (see recipe)
  • Screw‐cap microcentrifuge tubes, autoclaved
  • Microcapillary pipets
  • Densitometer (optional)
  • Additional reagents and equipment for tissue sample preparation (unit 10.2), agarose gel electrophoresis (unit 10.4) or ethidium bromide dot quantitation (unit 10.5), PCR (unit 10.20), Southern hybridization and UV cross‐linking (unit 10.6), and hybridizing blots with oligonucleotides (unit 10.6)
NOTE: Use sterile, distilled water to prepare all reagents. Do not use diethylpyrocarbonate (DEPC) to treat reagents. To avoid contamination with unwanted nucleic acids, prepare reagents and solutions solely for use in this protocol (see Critical Parameters). Wear disposable gloves and change them frequently.
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Figures

Videos

Literature Cited

Literature Cited
   Arrigo, S.J., Weitsman, S., Rosenblatt, J.D., and Chen, I.S.Y. 1989. Analysis of rev gene function on human immunodeficiency virus type 1 replication in lymphoid cells by using a quantitative polymerase chain reaction. J. Virol. 63:4875‐4881.
   Chory, J. and Baldwin, A.S. Jr. 1994. Nondenaturing polyacrylamide gel electrophoresis. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 2.7.1‐2.7.8. John Wiley & Sons, New York.
   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. U.S.A. 87:2725‐2729.
   Katz, J.P., Bodin, E.T., and Coen, D.M. 1990. Quantitative polymerase chain reaction analysis of herpes simplex virus DNA in ganglia of mice infected with replication‐incompetent mutants. J. Virol. 64:4288‐4295.
   Kwok, S., Mack, D.H., Mullis, K.B., Poiesz, B., Ehrlich, G., Blair, D., Friedman‐Kien, A., and Sninsky, J.J. 1987. Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection. J. Virol. 61:1690‐1694.
   Leib, D.A., Coen, D.M., Bogard, C.L., Hicks, K.A., Yager, D.R., Knipe, D.M., Tyler, K.L., and Schaffer, P.A. 1989. Immediate‐early regulatory gene mutants define different stages in the establishment and reactivation of herpes simplex virus latency. J. Virol. 63:759‐768.
   Li, H., Gyllensten, U.B., Cui, X., Saiki, R.K., Erlich, H.A., and Arnheim, N. 1988. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature (Lond.) 335:414‐417.
   Paabo, S. 1989. Ancient DNA: Extraction, characterization, molecular cloning and enzymatic amplification. Proc. Natl. Acad. Sci. U.S.A. 86:1939‐1943.
   Pang, S., Koyanagi, Y., Miles, S., Wiley, C., Vinters, H.V., and Chen, I.S.Y. 1990. High levels of unintegrated HIV‐1 DNA in brain tissue of AIDS dementia patients. Nature (Lond.) 343:85‐89.
   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 239:487‐488.
   von Beroldingen, C.H., Blake, E.T., Higuchi, R., Sensabaugh, G.F., and Erlich, H.A. 1989. Applications of PCR to the analysis of biological evidence. In PCR Technology: Principles and Applications for DNA Amplification (H.A. Erlich, ed.) pp. 209‐223. Stockton Press, New York.
Key Reference
   Katzet al., 1990. See above.
  Uses the protocol outlined here and presents examples of data generated.
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