Pcr Primer Design

Well-designed primers are probably the most important components of a good PCR reaction. The target region on the DNA template is defined by the position of the primers. PCR yield is directly affected by the annealing characteristics of the primers. For the PCR to work efficiently, the two primers must be specific to the target region, possess similar annealing temperatures, not interact significantly with each other or themselves to form 'primer dimers', and be structurally compatible. Likewise, the sequence region to which the primers bind must be fairly well conserved because if the sequence changes from one DNA template to the next then the primers will not bind appropriately. The general guidelines to optimal PCR primer design are listed in Table 4.4 (see also Dieffenbach et al. 1993).

A number of primer design software packages are commercially available including Primer Express (Applied Biosystems, Foster City, CA) and Oligo (Molecular Biology Insights, Cascade, CO). These programs use thermo-dynamic 'nearest neighbor' calculations to predict annealing temperatures and


Optimal Values

Primer length

Primer Tm (melting temperature)

Percentage GC content

No self-complementarity (hairpin structure)

No complementarity to other primer (primer dimer)

18-30 bases

< 3 contiguous bases

< 3 contiguous bases (especially at the 3'-ends)

< 2000 bases apart

Best match in BLAST search

Distance between two primers on target sequence Unique oligonucleotide sequence Tm difference between forward and reverse primers in pair <5°C No long runs with the same base < 4 contiguous bases aBLAST search examines similarity of the primer to other known sequences that may result in multiple binding sites for the primer and thus reduce the efficiency of the PCR amplification reaction. BLAST searches may be conducted via the Internet: http://www.ncbi.nlm.nih.gov/BLAST.

primer interactions with themselves or other possible primers (Mitsuhashi 1996, SantaLucia 1998).

The Internet has become a valuable resource for tools that aid primer selection. For example, a primer design program called Primer 3 is available on the World Wide Web through the Whitehead Institute (http://www.genome.wi.mit.edu/ cgi-bin/primer/primer3_www.cgi). With Primer 3, the user inputs a DNA sequence and specifies the target region within that sequence to be amplified. Parameters such as PCR product size, primer length and desired annealing temperature may also be specified by the user. The program then ranks the best PCR primer pairs and passes them back to the user over the Internet. Primer 3 works well for quickly designing singleplex primer pairs that amplify just one region of DNA at a time.

Table 4.4

General guidelines for PCR primer design.


The polymerase chain reaction permits more than one region to be copied simultaneously by simply adding more than one primer set to the reaction mixture (Edwards and Gibbs 1994). The simultaneous amplification of two or more regions of DNA is commonly known as multiplexing or multiplex PCR (Figure 4.3). For a multiplex reaction to work properly the primer pairs need to be compatible. In other words, the primer annealing temperatures should be similar and excessive regions of complementarity should be avoided to prevent

Figure 4.3

Schematic of multiplex PCR. A multiplex PCR makes use of two or more primer sets within the same reaction mix. Three sets of primers, represented by arrows, are shown here to amplify three different loci on a DNA template (a). The primers were designed so that the PCR products for locus A, locus B, and locus C would be different sizes and therefore resolvable with a size-based separation system (b).

Locus B

Locus C

->■ large the formation of primer-dimers that will cause the primers to bind to one another instead of the template DNA. The addition of each new primer in a multiplex PCR reaction exponentially increases the complexity of possible primer interactions (Butler et al. 2001).

Each new PCR application is likely to require some degree of optimization in either the reagent components or thermal cycling conditions. Multiplex PCR is no exception. In fact, multiplex PCR optimization is more of a challenge than singleplex reactions because so many primer-annealing events must occur simultaneously without interfering with each other. Extensive optimization is normally required to obtain a good balance between the amplicons of the various loci being amplified (Kimpton et al. 1996, Markoulatos et al. 2002).

The variables that are examined when trying to obtain optimal results for a multiplex PCR amplification include many of the reagents listed in Table 4.2 as well as the thermal cycling temperature profile. Primer sequences and concentrations along with magnesium concentrations are usually the most crucial to multiplex PCR. Extension times during thermal cycling are often increased for multiplex reactions in order to give the polymerase time to fully copy all of the DNA targets. Obtaining successful co-amplification with well-balanced PCR product yields sometimes requires redesign of primers and tedious experiments with adjusting primer concentrations.

Primer design for the short tandem repeat (STR) DNA markers that are discussed in Chapter 5 include some additional challenges. Primers need to be adjusted on the STR markers to achieve good size separation between loci labeled with the same fluorescent dye. In addition, the primers must produce robust amplifications with good peak height balance between loci as well as specific amplification with no non-specific products that might interfere with proper interpretation of a sample's DNA profile. Finally, primers should produce a maximal non-template-dependent '+A' addition to all PCR products (see Chapter 6).

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