Quantitation of Rare DNAs by PCR

Donald M. Coen1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 15.7
DOI:  10.1002/0471142727.mb1507s56
Online Posting Date:  November, 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, which can seriously affect the outcome of the procedure.

     
 
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Table of Contents

  • Reagents and Solution
  • Commentary
     
 
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Materials

Basic Protocol 1:

  Materials
  • 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)
  • 24:1 chloroform/isoamyl alcohol (unit 2.1)
  • 10 M ammonium acetate ( appendix 22)
  • Cold 100% ethanol
  • 70% ethanol
  • TE buffer, pH 7.5 ( appendix 22)
  • recipeReaction mix cocktail (see recipe)
  • Mineral oil
  • 0.8 U/µl Taq DNA polymerase (unit 15.1)
  • recipeOligonucleotide primers for hybridization (see recipe)
  • Screw‐cap microcentrifuge tubes, autoclaved
  • Microcapillary pipets or positive displacement pipets with disposable tips and plungers or micropipettors with barrier tips
  • Additional reagents and equipment for PCR (unit 15.1), tissue sample preparation (unit 2.2), agarose and nondenaturing polyacrylamide gel electrophoresis (units 2.5 & 2.7), ethidium bromide dot quantitation (unit 2.6), Southern blotting or electroblotting (unit 2.9), labeling oligonucleotides (units 3.10 4.6 & 4.8), hybridizing blots with oligonucleotides (units 2.9 & 6.4), UV cross‐linking DNA to filters (unit 2.9), and autoradiography ( 3.NaN)
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 and Troubleshooting). 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.
   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 hx (H.A. Erlich ed.) pp. 209‐223. Stockton Press (W.H. Freeman), New York.
Key Reference
   Katz et al., 1990. See above.
  Uses the protocol outlined here and presents examples of data generated.
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