Overview of PCR

Christine D. Kuslich1, Buena Chui2, Carl T. Yamashiro3

1 Molecular Profiling Institute, Phoenix, Arizona, 2 GE Healthcare, Piscataway, New Jersey, 3 Arizona State University, Tempe, Arizona
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 10.2
DOI:  10.1002/9780470089941.et1002s00
Online Posting Date:  October, 2008
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As a means of rapidly copying and amplifying a selected template sequence from a pool of DNA in vitro, the polymerase chain reaction (PCR) as a stand‐alone technique and in combinations with other methods has a vast range of applications. This chapter provides an overview of the theory and applications for this powerful and versatile laboratory method. A generic protocol for the broadest application is described in this unit along with the basic theory underpinning PCR to foster an understanding of how to make modifications to the protocol that can be applied to specific applications of the PCR technique. There is a troubleshooting table provided to describe and resolve the most common problems associated with PCR, as well as a table for suppliers and manufacturers of thermal cycling instruments. A detailed discussion of various DNA polymerases and suppliers is also provided.

Keywords: polymerase chain reaction (PCR); dNTPs; thermal cycler; DNA polymerase; DNA template; denaturation; annealment; elongation; melting temperature (Tm); hot start; primer design; contamination

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Table of Contents

  • Overview and Principles
  • Strategic Planning
  • Safety Considerations
  • Protocols
  • Basic Protocol 1: Routine PCR
  • Support Protocol 1: Using Temperature Gradients for Rapid Optimization of PCR Cycling Conditions
  • Support Protocol 2: Titration of MgCl2 Concentration
  • Reagents and Solutions
  • Understanding Results
  • Troubleshooting
  • Variations
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Routine PCR

  • 10% (w/v) bleach solution
  • Master mix components (see Table 10.2.5):
    • 5 U/µl DNA polymerase (e.g., Taq)
    • 10× PCR buffer with MgCl 2 (e.g., Promega, no. M8295; also see recipe) or without MgCl 2 (e.g., Promega, no. M3005; also see recipe); typically optimized for DNA polymerase of choice and provided by the manufacturer of the enzyme
    • 25 mM MgCl 2 (if not already included in PCR buffer)
    • 40 mM dNTPs (dATP, dTTP, dCTP, and dGTP; e.g., Promega, no. C1141; also see recipe)
    • 10 µm forward (upstream) and 10 µM reverse (downstream) primer (see recipe for Primers; custom synthesis available from Invitrogen)
    • Molecular‐biology‐grade, sterile, nuclease‐free ddH 2O (e.g., Invitrogen, no. 10977‐015)
  • DNA template (unit 5.2)
  • Sterile, nuclease‐free mineral oil (e.g., Sigma, no. M5904; only necessary if thermal cycler does not have heated lid)
  • Gel loading buffer/dye
  • Agarose gel
  • 100‐bp PCR ladder (e.g., Sigma, no. D3687)
  • Ethidium bromide
  • 0.2‐ml (e.g., VWR cat. no. 10011‐802) or 0.5‐ml (e.g., VWR, no. 10011836) thin‐walled reaction tubes (size dependent on thermal cycler block and manufacturer specifications)
  • Dedicated pipets used only for setting up PCR reactions (2 µl, 20 µl, 200 µl, and 1000 µl)
  • Sterile micropipettor tips (made for the pipets used for PCR reaction set up) with aerosol barrier (e.g., Rainin Instruments)
  • Vortex
  • Thermal cycler (see Table 10.2.2 for a list of manufacturers)
  • UV transilluminator
  • Additional reagents and equipment for preparing the DNA template (unit 5.2) and agarose gel electrophoresis including staining with ethidium bromide (unit 7.2)
NOTE: The time to complete reaction preparation will vary depending on the number of samples to be run. Typically setting up ten samples will take 30 to 45 min.
Table 0.2.5   MaterialsStandard PCR Reaction Mixture

Components Final concentration Per tube volume Master mix for 10 tubes (prepare for 12 tubes)
10× PCR buffer MgCl 2‐free 5 µl 60 µl
25 mM MgCl 2 a 1.5 mM 3 µl 36 µl
40 mM dNTP mix b 0.2 mM each dNTP 1 µl 12 µl
10 µM forward primer 1 µM c 5 µl 60 µl
10 µM reverse primer 1 µM c 5 µl 60 µl
Sterile, nuclease‐free H 2O 30.75 µl (to a final volume of 50 µl) 369 µl
5 U/µl hot‐start Taq DNA polymerase d 0.025 U/µl 0.25 µl 3 µl
>100 ng/µl template e 2 ng/µl 1 µl

 aMgCl 2 concentration can be titrated to optimize the PCR reaction. MgCl 2 concentration ranges between 1.5 and 4.0 mM for most PCR.
 bThere are numerous commercially available dNTP mixes. Most are 40 mM (10 mM of each of the 4 dNTPs).
 cPrimer concentration can also be titrated to optimize the PCR reaction. Final primer concentrations generally can be adjusted within the range of 0.2 to 1 µM.
 dConcentrations of Taq will vary. When using a non‐hot‐start polymerase, the enzyme should not be added to the reaction vessel until the tubes are at 95°C in the thermal cycler to avoid the formation of nonspecific products.
 eTemplate DNA concentration can vary, but generally the final concentration of the template DNA will not exceed 10 ng/µl. The technique is sensitive enough to detect pmol quantities of template.

Support Protocol 1: Using Temperature Gradients for Rapid Optimization of PCR Cycling Conditions

  • Thermal cycler with temperature gradient function
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Key References
   Mullis et al., 1986. See above.
  The three key references provided above are historical in nature and illustrate some of the noteworthy efforts made early on in the development of this prevalent technique.
   Saiki et al., 1985. See above.
   Saiki et al., 1988. See above.
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