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Overview of PCR

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

¹Molecular Profiling Institute, Phoenix, Arizona
²GE Healthcare, Piscataway, New Jersey
³Arizona 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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Abstract

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 (T_m); hot start; primer design; contamination

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Overview and Principles
Strategic Planning
Safety Considerations
Protocols
Basic Protocol: 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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Materials

Basic Protocol: Routine PCR

Materials

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 MgCl₂ (e.g., Promega, no. M8295; also see recipe) or without MgCl₂ (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 MgCl₂ (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 ddH₂O (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 10.2.5 Standard PCR Reaction Mixture

Components	Final concentration	Per tube volume	Master mix for 10 tubes (prepare for 12 tubes)

10× PCR buffer MgCl₂-free	1×	5 µl	60 µl
25 mM MgCl₂^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 H₂O		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	—

^aMgCl₂ concentration can be titrated to optimize the PCR reaction. MgCl₂ 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

Additional Materials (also see Basic Protocol)

Thermal cycler with temperature gradient function

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Figures

Figure 10.2.1

PCR process. Depiction of first cycle showing the denaturation of the target sequence using high temperature (92° to 96°C), primer (short gray segment) annealing (e.g. at 58°C), and elongation by DNA polymerase (denoted with P; e.g. at 72°C). Subsequent cycles will double the number of starting amplicons for that cycle.

View Image
Figure 10.2.2

Inhibition of nonspecific product amplification using a hot-start PCR system. Amplification of a 1018 bp fragment (lanes 1 through 8) and a 300 bp fragment (lanes 9 through 16), randomly selected sequences within the human genome. PCR reactions with illustra Hot-Start Mix RTG are shown in lanes 1 through 4 and 9 through12, PCR reactions with standard Taq DNA polymerase, a non-hot-start system, are shown in lanes 5 through 8 and 13 through 16. In all reactions, 10 ng of human genomic DNA was used with 10 pmol of forward and reverse primers. The products and DNA size markers (M) were resolved on a 1.5% agarose TAE gel stained with ethidium bromide. In lanes 5 to 8, there are low molecular weight bands of which the lowest could be a primer dimer (unconfirmed); however, in the hot-start reactions, it is clear that nonspecific binding events were prevented, successfully leading to efficient, specific single product amplification of the expected size. Data and image used with permission from GE Healthcare.

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Figure 10.2.3

Thermal cyclers. Representative thermal cyclers within research laboratories: (A) thermal cycler (Applied Biosystems) with a single peltier block holding up to 96 tubes or a 96-well plate; and (B) thermal cycler (Bio-Rad Laboratories) with four independently controlled peltier blocks, each holding up to 96 tubes or a 96-well plate.

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Figure 10.2.4

UNG contamination control process. PCR is performed normally except that dUTP replaces dTTP in the reaction, as well as inclusion of the UNG enzyme. The resulting amplicons will have incorporated dUTP. The dUTP will not affect downstream analysis of the amplicons (e.g. visualization of the amplicons by agarose electrophoresis). However, if the dU-containing amplicons contaminate a subsequent PCR that has UNG present, then during the UNG pre-treatment incubation any dU will be modified to render it unamplifiable in the PCR. The UNG enzyme is inactivated by high temperature treatment (usually coincides with the first cycle denaturation and Hot-Start enzyme activation if used) and therefore will not be able to degrade the newly created amplicons in the subsequent PCR reaction. PCR will only amplify natural DNA targets containing dT rather than dU.

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Figure 10.2.5

Thymidine dimer contamination. Standard PCR is performed. Prior to post-PCR analysis, the reaction mix is exposed to short-wave UV light to form thymidine (T-T) dimers which cannot participate in future PCRs should these amplicons contaminate a new PCR reaction. Black bars denote T-T dimer formation.

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Figure 10.2.6

Psoralen contamination-containment procedure. Standard PCR with the inclusion of psoralen (or isopsoralen; shown as open circles) is run. Upon exposure of the completed reaction to UV light, psoralen will conjugate to dT bases (a T within a circle). The attached psoralen will inhibit any replication by DNA polymerases and thus is no longer a potential interfering contaminant for future PCRs.

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Figure 10.2.7

Laboratory design for contamination containment. (A) Pre-PCR set up laboratory with an anteroom; storage cabinets for materials and supplies; 4°C refrigerator and –20°C freezer for storage of enzymes, samples and other items requiring cold storage; workstations for setting up PCRs in a contained environment; and air pressure regulation for laboratory spaces: (++) denotes positive pressure, (+) denotes slightly less positive pressure, and (0) denotes normal ambient laboratory air pressure. (B) Thermal cycling and post-PCR analysis laboratory is typically not adjacent to the pre-PCR set up laboratory; contains thermal cycler(s), equipment for running and visualizing (including photo documentation) gels, cold storage for reagents, supplies and storing completed PCRs, and an area for other post-PCR analytical activities (i.e. sequencing or hybridization studies); and notation of laboratory air pressure, with the laboratory having negative pressure, (–) relative to the main laboratory area with standard ambient pressure.

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Figure 10.2.8

Understanding PCR results. An example of gel electrophoresis of a PCR for a given gene using a 1.5% agarose gel (unit 7.2). Primers were designed to flank a region known to have insertion and deletion mutations. Lane M has a DNA ladder loaded (various sized DNA bands are pointed out in the figure). Lane 1 is an example the normal band (680 bp). Lane 2 shows both the normal 680 bp product and the presence of a smaller 517 bp deletion product. Lane 3 shows both the 680 bp amplicon and the presence of a larger 1033 bp insertion product. Lane 4 (labeled NTC) is the no-template control reaction run with all of the reagents used in the PCR reaction (the remaining portion of the master mix) except the template. The absence of any amplification in the NTC demonstrates that there was no contaminating template in the reagents master mix that would account for the amplification seen in lanes 1 to 3.

View Image

Literature Cited

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Key References
	Mullis et al., 1986. See above.
	Saiki et al., 1985. See above.
	Saiki et al., 1988. 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.