CHAPTER ONE

Explanatory Chapter: PCR

Primer Design
Rubén Álvarez-Fernández1
Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom
1
Corresponding author: e-mail address: [Link]@[Link]

Contents
1. Theory 2
1.1 Thermodynamics 2
1.2 General guidelines for primer design 3
1.3 Melting temperature (Tm) and GC content 3
1.4 Primer length 4
1.5 Primer sequence and secondary structures 5
1.6 PCR product length and placement within the target sequence 6
1.7 Step-by-step guide for primer design 7
1.8 Troubleshooting and general rule 8
1.9 Specific applications 8
1.10 Target cloning 8
1.11 Reverse-transcription PCR (RT-PCR) 9
1.12 Real-time PCR (quantitative PCR, qPCR) 10
1.13 Multiplex PCR 12
1.14 Sequencing 12
1.15 Degenerate primers 13
1.16 Modified primers 16
1.17 Recombineering 16
1.18 Primers for homologous recombination 17
1.19 Software tools list 18
Acknowledgments 19
References 19

Abstract
This chapter is intended as a guide on polymerase chain reaction (PCR) primer design
(for information on PCR, see General PCR and Explanatory Chapter: Troubleshooting
PCR). In the next section, general guidelines will be provided, followed by a discussion
on primer design for specific applications. A list of recommended software tools is
shown at the end.

Methods in Enzymology, Volume 529 # 2013 Elsevier Inc. 1

ISSN 0076-6879 All rights reserved.
[Link]
2 Rubén Álvarez-Fernández

1. THEORY
In the spring of 1983, Kary Mullis had the idea to use a pair of synthetic
oligonucleotides (primers) to potentially copy ad infinitum a target DNA
sequence using DNA polymerase; an idea worthy of a Nobel Prize. How-
ever, thermal cycling was the essence of the process, and posed a critical
drawback: the double-stranded DNA was denatured by heat at the begin-
ning of each cycle, destroying the polymerase in the process, and it had
to be replaced in each round. In 1986, Mullis solved the problem by using
a thermo-resistant DNA polymerase from Thermophilus aquaticus (Taq).
With the drawback gone, the PCR became dramatically more affordable
and was subject to automation.
Now, PCR is a core technique in molecular biology and an extremely
large number of applications have been developed. These methods directly
depend on the efficiency of the reaction, which relies on the fine-tuning of
its components: DNA template, dNTPs, reaction buffer, and a set of primers
that flank the target sequence and are extended by the DNA polymerase.
This chapter focuses on primer design, a critical factor to both the effi-
ciency and the specificity of the PCR.

1.1. Thermodynamics
The changes in two thermodynamic properties of a system (DS – change of dis-
order, and DH – change of heat) can be used to calculate DG, the Gibb’s free
energy change. This value shows whether a process is spontaneous, and allows
one to determine the equilibrium constant K, with which the concentrations of
all the species that are involved in the equilibrium can be worked out. There-
fore, the thermodynamic changes that happen in going from random coil to
duplex can be known and used to determine the primer quality. A full thermo-
dynamic explanation is given in SantaLucia (2007); Mann et al. (2009).
To quickly sum up, the DG is directly related to the equilibrium constant
(K) of the primer/template hybridization at a given temperature (T).
DG ¼ DH  T DS
K ¼ ½primer plus template duplex=ð½primer½templateÞ
DG ¼  RT  lnðK Þ
A key parameter that affects the efficiency of a PCR is the amount of
primer bound to the template. If the concentration of duplex (primer bound
Explanatory Chapter: PCR Primer Design 3

to template) is different for each of the primers, the efficiency will be

reduced. From the formulae above, it can be deduced that designing primers
with the same DG (at a fixed annealing temperature) will render more effi-
cient primers pairs, and also explains why matching Tm’s is a less accurate
approach than matching DG’s.
The more negative the DG the higher the K, and then the more stable
the duplex. Primer software sets thresholds that are used to discard bad
primers, as secondary and tertiary structures (primer-dimers, hairpins,
etc.) with very low DG (very stable) are undesirable.

1.2. General guidelines for primer design

Primer design has two goals: specificity and efficiency of amplification.
Specificity is the frequency of proper priming events – mispriming leads
to amplifications off-target, though mindfully used it can serve to hunt
for related sequences (e.g., degenerate primers, see below). Efficiency is
the increase of product in each PCR cycle; the theoretical optimum is a
two-fold increase. The application determines the balance between these
parameters. For instance in diagnostics, efficiency is sacrificed for a higher
specificity, minimizing the false positives at a cost of less PCR product.
These two parameters are controlled by factors such as temperature, exten-
sion time, template concentration and thermodynamic properties, length of
the amplicon, divalent cations (Mg2þ, Mn2þ), and detergents.
The main parameters affecting primer design and therefore the PCR
reaction are discussed below.

1.3. Melting temperature (Tm) and GC content

Tm is the temperature at which half the primer strands are bound to the tar-
get. It depends on base composition and can be roughly estimated as
Tm ¼ 4ðG þ C Þ þ 2ðA þ T Þ
However, this equation neglects the dependence of Tm on strand con-
centration, salt concentration, and base sequence. Typical errors for this
method compared with determining the Tm experimentally can be
>15  C, thus this equation is not recommended (SantaLucia, 2007).
The best current approach to calculate Tm is the nearest-neighbor (NN)
method (SantaLucia, 1998). The previous 1986 NN model is unreliable, but
still present in most common primer design tools such as Primer3, OLIGO,
4 Rubén Álvarez-Fernández

and Vector NTI®. When possible, then, the 1998 model should be selected
(SantaLucia, 2007).
Tm depends on base content. Two primers with a different length can
have the same Tm then, but the longer one will anneal less efficiently than
its partner, thereby affecting the efficiency of the PCR. A different Tm
between the pair will affect specificity (at lower annealing temperatures,
the primer with higher Tm will be more prone to misprime) and efficiency
(at higher temperatures, the primer with lower Tm will anneal less frequently
or not at all). DHo of binding is different for each primer, resulting in dif-
ferent slopes of binding at the Tm, and then in different amplification effi-
ciencies and artifacts. Therefore, the key point is not to match Tm, but equal
amounts of primers bound to the target: to do so, design primers with
matching DGo at the desired annealing temperature (SantaLucia, 2007).
Typically, the annealing temperature to start with is 4–10  C below the
Tm. However, the optimal annealing temperature can be more accurately
calculated as
Ta ¼ 0:3Tm,primer þ 0:7Tm,product  14:9
with Tm,primer being the Tm of the less stable primer-template pair
(Rychlik, 1990. Note that Tm calculations are not made with the latest
NN model – SantaLucia, 1998).
In summary, for regular PCR, a match of DGo (otherwise no more than
2  C of difference in Tm) and GC content (40–60%) is advised. However,
the GC content can be higher and work fine, so the more general rule is that
it should be not be lower than that of the amplicon (Rychlik, 1993).

1.4. Primer length

A major factor controlling specificity is the annealing temperature, which is
determined by the length and base composition of the primers.
The shorter the primer, the quicker the annealing with the template and
the lower the specificity, therefore, primers <18 nucleotides in length are
likely to perform badly with complex templates (e.g., genomic DNA).
Primers 16–18 nucleotides in length will perform well only for <500 bp
amplicons; for longer amplicons, longer primers are recommended, for
example, 24 nt primers will do for 5 kb products (Rychlik, 1993).
The longer the primer, the lower the efficiency (a small inefficiency will
propagate with the number of cycles). However, a longer primer leads to a
higher specificity because of the primer length (4 times per added base) and
Explanatory Chapter: PCR Primer Design 5

its melting temperature. Thus, 28–35 nt primers can be used to amplify tem-
plates expected to have a high degree of heterogeneity, or for cloning closely
related genes. It is important to note here that longer primers are less pure
due to inefficiencies in chemical synthesis, so purification better than stan-
dard desalting might be required for certain applications (see Purification of
DNA Oligos by Denaturing Polyacrylamide Gel Electrophoresis (PAGE)).
In summary, the utilization of primers of a minimal length that ensures
Tm >54  C is recommended to balance specificity and efficiency in general
conditions (Dieffenbach et al., 1993).

1.5. Primer sequence and secondary structures

The primer’s sequence determines its specificity for the target but can also
facilitate interaction with itself and other primers, affecting its performance.
Taq polymerase extends only the 30 -end of the primer, so its sequence is
critical. Perfect base pairing for at least the last 4 bases is optimal, and minimal
mismatch should exist within the last 5–6 ones (lowering the annealing tem-
perature does not compensate well for mismatches near the 30 -end of the
template, but rather it promotes mispriming). Conversely, the 50 -end
sequence is not that critical, so 50 tails (e.g., restriction sites, fluorophores,
etc.) can be added to the primers without significantly affecting
annealing efficiency.
Primers with low 30 -end stability (AT-rich, DG>9 kcal mol1) but a
stable 50 -end perform best in sequencing and PCR, since this structure
reduces mispriming (Rychlik, 1993). Therefore, structures with high 30 -
end stability such as GC clamps (more than 3 G or C bases within the last
five 30 -end bases) should be avoided because the stronger bonding raises
the likelihood of partial priming.
Avoid also mononucleotide runs longer than 4 nucleotides (e.g., GGGG)
and repeats of more than 4 dinucleotides (e.g., ATATATAT), because they
can cause mispriming and polymerase slippage (see Viguera et al., 2001 for
more information on slippage).
In general, discard primers that form intramolecular or primer/primer
duplexes with negative DG, especially if these structures are formed at the
30 -end. Although Tm,duplexes <Tm,primer/template dimerization will occur to
some extent, lowering the PCR efficiency by sequestering the polymerase
and the primers themselves. For instance, hairpins can cause internal primer
extension if they form at the 30 -end, eliminating that primer from the reac-
tion (though on the 50 -end do not significantly affect the PCR). Primer-
6 Rubén Álvarez-Fernández

dimers are extended by the polymerase and can be detected on an agarose gel
as a low molecular weight band, or on a melting curve (usually performed in
qPCR experiments) as extra peaks. ‘Fake’ primer-dimers can also appear if
the primers bind off-target close to one another, which explains why some-
times the band is longer than expected, and why sequencing shows a few
extra unexpected bases between the actual primers.
Prevention of dimerization is crucial in qPCR experiments and multi-
plex PCR. Patches like the hot start method can mitigate it but, if possible,
it is preferable to avoid it from the beginning.
An approximation that meets these problems is the proprietary
ThermoBLAST™, by DNA software, which can be implemented into their
Visual OMP™ primer design package. Although a BLAST search will give
information on mispriming, it just considers base pairing and not thermody-
namic parameters like duplex stability, so its help is limited. ThermoBLAST™
retains the computational efficiency of BLAST but uses thermodynamic scor-
ing for base pairs, dangling ends, single mismatches, bulges, tandem mis-
matches, and other motifs, thereby rendering more balanced primer pairs
(SantaLucia, 2007). Pythia is another software approach in which state-of-the
art DNA binding affinity computations are directly integrated into the primer
design process. It seems it is comparable to other available software, although it
outperforms them on the design for primers on difficult regions (e.g., repeats).
In summary, select those primers less likely to form secondary structures
and with low 30 -end stability.

1.6. PCR product length and placement within the

target sequence
The product length affects the efficiency of the PCR. Generally, 150–
1000 bp products are amplified for target detection, and some techniques
like qPCR (see below, real-time PCR) have narrower windows for the length
of the amplicon. For detection of gene expression, it is preferable to design
the primers on contiguous exons (see below, RT-PCR).
If possible, avoid regions with long runs, repeats, or secondary structures,
because they will make good primer design and matching more compli-
cated. Those structures can be predicted with software such as UNAFold
(also called Mfoldþþ). Some primer design software, such as Phytia, takes
into consideration binding and folding energies, outperforming other soft-
ware for designing primers for repeat regions. Proprietary software like
Visual OMP™ also allows for prediction of secondary structures and can
solve the multistate coupled equilibrium of these cases (SantaLucia, 2007).
Explanatory Chapter: PCR Primer Design 7

1.7. Step-by-step guide for primer design

These are some guidelines for RT-PCR primer design, but can mostly be
applied to design primers for other purposes.
Conventions:
• Sequences are always written from 50 to 30 , no matter whether they are
forward or reverse primers (therefore, do not ever write a primer sequence
reversed because it will lead to confusion).
• Polymerase extends from the 30 -end.
Procedure:
1. Get the cDNA sequence. If possible, use RefSeqs.
Refseq, or Reference Sequence database from NCBI, is a collection
of annotated DNA, RNA, and protein sequences. Each RefSeq is
reviewed and represents a single molecule from one organism (http://
[Link]/RefSeq/). Sequences other than RefSeqs do
not have that assured quality and there is a chance they are truncated,
untrimmed, or incorrect. Make sure you are using the latest version
of a RefSeq entry (i.e., the accession with the highest version number),
or simply just use the accession without the version number, as you will
automatically get the latest version.
2. Primers can be designed from the NCBI itself by clicking on ‘pick
primers,’ next to the sequence. The tool combines Primer3 and BLAST
and is called Primer-BLAST.
Primer-BLAST allows one to design primers bridging an exon–exon
junction or spanning an intron (see below for explanation, on RT-PCR),
thereby avoiding false positives due to DNA amplification. The BLAST
addition allows for the selection of organism-specific primers and selec-
tive amplification of splice variants.
3. Select the parameters that suit your specificity goals (see guidelines
above) and the software will provide the best primer pairs (Note:
Primer-BLAST uses the 1998 model for Tm calculation by default).
4. Primer quality can be double-checked using software such as
NetPrimer.
5. Alternatively, design the primers using software like Primer3,
Primer3Plus, Pythia, Visual OMP™, OLIGO, or Vector NTI®
(just a few examples in no particular order. The first 3 are freely available
and the last 3 are proprietary).
6. Finally, for RT-PCR, it is recommended that primers be chosen close to
the mRNA 30 -end. The reverse transcription reaction starts at this end,
and premature termination of the reaction can produce shorter
8 Rubén Álvarez-Fernández

molecules that lack the 50 -end. Specially if poly-(T) are used, designing
the primers close to the 30 -end is suggested to avoid the underestimation
of the target concentration.

1.8. Troubleshooting and general rule

If no specific product is achieved in the first attempt, alternative approaches can
be used which are, however, beyond the scope of this chapter (see Explanatory
Chapter: Troubleshooting PCR). A gradient PCR is very useful to determine
empirically the best annealing temperature for the primer set. A touchdown
PCR will increase specificity at a cost of lower efficiency. A nested PCR will
enrich the sample in specific product. The use of a different polymerase can dra-
matically affect the results as well (e.g., switching from regular Taq to a high-
fidelity polymerase). Amplification of some DNA templates works better at
extension temperatures lower than 72  C (especially if they are <500 bp or
highly AT-rich, and therefore less stable at high temperatures). The use of
two-step PCRs (denaturation plus annealing-extension, by designing primers
with a Tm of 68–70  C) or tweaking the PCR mix (changing the Mg2þ con-
centration, addition of DMSO, etc.) can also help. See Mülhardt (2007) for fur-
ther information on PCR troubleshooting.
However, the general rule can be: begin with well-designed primers, and
if a few troubleshooting attempts fail, consider designing a new set of primers
instead of trying to tackle all of the modifications possible.

1.9. Specific applications

Ultimately, primer design strategy is determined by the goal of the PCR
method. Several designs are discussed below; it is important to note that
PCR depends not only on primer design, but also on primer quality and
purity. Chemical synthesis of primers ends up with a salty mixture of oligo-
nucleotides of different lengths, or a mixture of labeled and unlabeled ones.
They have to be purified, and the purification method should be chosen in
terms of the final application. Therefore, a faulty PCR can happen after a
perfect primer design, simply because in the reaction we are adding inter-
fering faulty oligonucleotides. See Purification of DNA Oligos by Denatur-
ing Polyacrylamide Gel Electrophoresis (PAGE) for further explanation.

1.10. Target cloning

There are several approaches for cloning (see Molecular Cloning and Res-
trictionless cloning). Primers can be designed at the ends of a target and the
Explanatory Chapter: PCR Primer Design 9

PCR product cloned into an A/T vector (even directionally cloned by

doing some modifications, see Zhou and Gomez-Sanchez, 2000). This
can be done in Primer3Plus by choosing the option ‘cloning.’
Alternatively, restriction sites can be designed as 50 -tails in the primers,
where they do not substantially affect annealing (if necessary, lower the
annealing temperature for the first few cycles and then raise it to the
annealing temperature considering the whole primer including the tail).
Restriction enzymes will not cut efficiently if just the target restriction site
is added; adding 4–6 bases at the 50 -end are usually required for best perfor-
mance. New England Biolabs offers some information on this subject:
[Link]
enzymes/cleavage_linearized_vector.asp.
[Link]
enzymes/cleavage_olignucleotides.asp (bear in mind that these examples
are oligonucleotides. Therefore, only a few bases are needed at the
50 -end of the restriction site since the 30 -end is linked to the primer).
Check that the target sequence does not contain the desired restriction sites, design the
primers, and add the 50 -tails containing the restriction sites. If the sequence
contains the restriction site(s), you can still use different restriction enzymes
for insert and vector that generate compatible ends (e.g., Sal I and Xho I; see a
complete list at [Link]
tion_enzymes/compatible_cohesive_overhangs.asp).
For further reference on restriction enzymes, check the NEB website at
[Link]
[Link]?

1.11. Reverse-transcription PCR (RT-PCR)

RT-PCR (see Reverse-transcription PCR (RT-PCR)) is sensitive enough
to enable detection and quantitation of RNA from even a single cell. In con-
trast, two other commonly used techniques for quantifying mRNA levels –
Northern blot analysis (see Northern blotting) and nuclease protection assays
(see Explanatory Chapter: Nuclease Protection Assays) – require larger of
amounts of RNA.
A potential problem with RT-PCR is contamination of the RNA with
genomic DNA, which can result in false positives. Consequently, primers
should be designed to span introns or bridge an exon–exon junction
(Fig. 1.1). When they span one or more introns, the amplified product from
genomic DNA will be bigger than expected, or there will be no product at
all if the distance between the primers exceeds the DNA polymerase
10 Rubén Álvarez-Fernández

exon forward primer

intron reverse primer
3' partially annealed forward primer

Primers spanning introns

Template PCR product
exon 1 exon 2

exon 1 exon 2

exon 1 exon 2 No product

(DNApol not processive enough)

Primers bridging exon-exon junctions

exon 1 exon 2 exon 3

X No product
exon 1 exon 2 exon 3
(no priming)

exon 1 exon 2 exon 3

X partial priming!!
exon 1 exon 2 exon 3

Figure 1.1 Considerations on RT-PCR primer design. DNA contamination in RNA can
result in false positives. Consequently, primers should be designed to span introns or
bridge an exon–exon junction, so the cDNA-derived PCR product is distinguishable from
the genomic DNA-derived one. When bridging exon–exon junctions, take care not to
design a primer with too long a 30 -end or GC clamps which allow for extension, since
you will not be able to distinguish between cDNA- and genomic DNA-derived amplifi-
cations (example at the bottom).

processivity. When they bridge an exon–exon junction, they will not

amplify genomic DNA template because the intron will hinder the
annealing. Make sure you avoid GC clamps and that only a few nucleotides
from the 30 -portion of the primer can base pair with the 30 -exon, because a
substantial pairing can still prime the polymerase. See above Step-by-step guide
for primer design, where a RT-PCR-oriented designing protocol is provided.

1.12. Real-time PCR (quantitative PCR, qPCR)

(Adapted from [Link]
Guidelines for primers:
– Length: 18–30 nucleotides
– %GC: 30–80% (ideally, 40–60%)
Explanatory Chapter: PCR Primer Design 11

– Tm: 63–67  C (ideally, 64  C), so the annealing temperature would be

58–60  C (Note: as discussed above, matching DG is more accurate than
matching Tm. Otherwise, avoid Tm differences larger than 4  C).
– Avoid mismatches, runs, repeats, and self- and primer-primer
complementarity.
– Avoid a T residue at the 30 -end (allows mismatching).
– Make sure the primers are specific and target a single amplicon by BLAST.
– The considerations given in the section on RT-PCR should also be
observed if a RT-qPCR is to be performed.
– For qPCR, it is preferable to design primers bridging exon–exon
junctions (see Fig. 1.1), since these will amplify specifically the mRNA.
If there is genomic DNA contamination, primers spanning introns
will amplify the contaminant template and the resulting product will
interfere with the accurate quantification of the target. It is then rec-
ommended that a separate control be set up to detect genomic DNA
contamination.
Guidelines for probes:
– Length: 18–30 nucleotides (optimal length is 20 nt. If >30 nt are
required, use an internal quencher on dT around the 20th nucleotide
rather than at the 30 -end).
– %GC: 30–80%.
– Tm: 8–10  C higher than the Tm of the primers (8  C for genotyping,
10  C for expression profiling).
– Select the strand that gives the probe more Cs than Gs.
– Place the probe the close to the primers without overlapping them.
– Avoid mismatches, runs, repeats, and self- and probe-primer
complementarity.
– Avoid a G residue at the 50 -end because it quenches the fluorophore.
– Make sure the probe is specific and targets a single amplicon (the same as
the primers) by BLAST.
– If genotyping by multiplexing, position the polymorphism in the center of
the probe and match the probe’s DG if possible, otherwise match the Tm.
Guidelines for amplicons:
– Length for SYBR® Green I assays: 80–150 nucleotides (shorter amplicons
will increase the PCR efficiency, and longer ones will give a higher DRn
as more dye will be incorporated).
– Length for 50 exonuclease probe assays: 80–120 nucleotides (shorter
amplicons will increase the efficiencies of the PCR and 50 nuclease
reactions).
12 Rubén Álvarez-Fernández

– %GC: 30–80% (ideally, 40–60%).

– Avoid secondary structures in the amplicon (they can be checked with
UNAFold).
It is critical that the primers are well designed. No secondary structures and
very close DG’s, no annealing to parts of the template that can result in
unbalanced binding, so that the efficiency of amplification is close to
100% and no off-target amplification occurs.
See Explanatory Chapter: Quantitative PCR and Wong and Medrano
(2005) for further information regarding real-time PCR.

1.13. Multiplex PCR

The goal of multiplex PCR is to amplify several targets in the same tube.
Since first described by Chamberlain et al. (1988), it has had many applica-
tions and is typically used for genotyping, where simultaneous analysis of
multiple markers is required, for detection of pathogens or genetically mod-
ified organisms, or for microsatellite analyses. Multiplex assays are difficult to
establish, so they are worthwhile only for analyses that will be repeated many
times, since at the end, they save space, money, and time.
Two or more primer pairs are used at a time. They could be designed and
work separately, but when put all together, primers from one pair can inter-
act with primers from another one. This makes the optimization of multi-
plex PCR quite difficult, as each primer pair could have different
requirements. To match the Tm and DG is just not enough because of
the potential interactions, and an effort made in primer design is worthwhile.
Sets of primer pairs can be checked for cross-dimerization using tools like
AutoDimer.
If starting from scratch, there is software for multiplex PCR primer
design: MIPS is specially made for designing multiplex PCR experiments
for SNP genotyping, PrimerStation is specific for the human genome,
and the commercially available Visual OMP™ can deal with multistate
equilibrium reactions and multiplex design as well (SantaLucia, 2007).

1.14. Sequencing
As stated above, sequences are written from 50 to 30 and the polymerase
extends from the 30 -end. Sequencing is done on just one strand, so you have
to make sure you design the primer for the right one.
Additionally consider that whilst PCR is exponential, sequencing is not.
Therefore, bad primers that work for PCR after tweaking the reaction might
Explanatory Chapter: PCR Primer Design 13

not be suited for automated sequencing: mismatched primers will create

templates that are perfect matches in a PCR, allowing amplification, but that
will not happen with sequencing since only one strand is replicated.
Desired conditions:
• Use accurate sequence data, as mismatching will reduce the specificity
and efficiency of the sequencing reaction, leading to bad readings or
overlapped sequences. Verify the site-specificity of the primer. The last
8–10 bases at the 30 -end should be unique. If you repeatedly get over-
lapped sequences, primers might be partially annealing to the vector or
on a different place on the insert, and you should redesign them.
• Design the primers no <50 bases from the sequence you are interested in
(because the first reads are usually inaccurate), but not farther than 300
bases from it (usable sequence reads reach up to 700–1000 bases).
Sequence data is often most accurate about 80–150 nucleotides away
from the primer.
• Optimal primer length is 20–25 nucleotides (acceptable range is
18–30 nt).
• %GC content: 40–60%.
• Tm within the range of 55–65  C.
• Discard candidate primers that self-anneal
• Try to choose primers with <3 Cs or Gs at the last 5 bases at the 30 -end,
and that do not end with C or G. This will reduce the likelihood of par-
tial unspecific priming.
Some software, such as Primer3Plus, has a specific option for designing
sequencing primers.
Additionally, software has been developed specifically for directed
sequencing, such as JCVI Primer Designer. This particular software is
designed for pipelining high-throughput PCR primer design, and high-
quality directed sequencing results have been obtained using it (as a note,
this software is based on Primer3 but does not use the latest NN Tm calcu-
lation method of SantaLucia (1998)).
Anyway, always check with your sequencing facility as they might have
specific requirements for primer design, template concentration, etc.

1.15. Degenerate primers

A PCR primer sequence is called ‘degenerate’ if some of its positions have
several possible bases. The ‘degeneracy’ of the primer is the number of
unique sequence combinations it contains. Hence, these primers are
14 Rubén Álvarez-Fernández

supplied as mixtures of similar, but not identical, oligonucleotides. They

have various applications such as amplification or cloning of related genes
from the same or different organisms, cloning of genes based on protein
sequence, or genome walking with techniques such as TAIL-PCR (thermal
asymmetric interlaced PCR, Liu and Whittier, 1995).
Sequence comparison of multiple members of protein families has rev-
ealed protein motifs and domains that play important roles in protein func-
tion. Closely related sequences can be cloned using degenerate primers
(a pool of primers containing most, or all, possible nucleotide sequences
encoding a conserved amino acid motif ) or consensus primers (a single
primer containing the most common nucleotide at each codon position
within the motif ).
The goal is then to design primers that match as many sequences of
homologous genes as possible. A naı̈ve solution would be to align the
sequences without gaps, count the number of different nucleotides in each
position along the alignment, and seek a primer-length window (20–30 bp)
where the product of the count is low. Such a solution is insufficient because
of gaps, the inappropriate objective function of the alignment, and most
notably, the exceedingly high degeneracy: when degeneracy is too high,
unrelated sequences may be amplified as well, losing specificity (Linhart
and Shamir, 2002; 2005). The degeneracy has to be high to maximize
the coverage, but has to be bound to decrease the probability of amplifying
unrelated sequences. This problem and the approaches to solve it are
addressed in Najafabadi et al. (2008).
Below, some software approaches are suggested for the design of degen-
erate primers:
PAMPS (Pairwise Alignment for Multiple Primer Selection). In con-
trast to previous algorithms, this one does not restrict the output to the exact
primer length that was given, which allows selecting an appropriate primer
in terms of annealing temperature. PAMPS can be used to design degenerate
primers for amplifying genes with uncertain sequences, such as new mem-
bers of gene families or libraries of antibody variable fragments. PAMPS has
been shown to outperform previous algorithms (HYDEN and MIPS) in this
task. This software is freely available.
Other tactics have been developed based on COnsensus-DEgenerate
Hybrid Oligonucleotide Primers (CODEHOPs). A CODEHOP is a
hybrid primer consisting of a 15–20 nt consensus 50 -clamp and a 9–12 nt
degenerate 30 -core region. The core gives a broader specificity for distantly
related target gene templates, while the clamp allows for a robust
Explanatory Chapter: PCR Primer Design 15

amplification during the later cycles of the PCR. CODEHOP works well
for small sets of proteins, taking into account the codon usage of the target
genome and the desired annealing temperature. However, it is inappropriate
for constructing primers with very high degeneracy on large sets of long
genomic sequences, and software like PAMPS should be used for that task.
An interactive version of the software, iCODEHOP, is freely available.
MAD-DPD (Minimum Accumulative Degeneracy Degenerate Primer
Design). MAD-DPD can be considered as a first approach in primer design
when a high degeneracy is desired and the number of degenerate primers to
be constructed is known or has to be kept low. A drawback of this software is
that it does not consider parameters like melting temperature, self-annealing,
or secondary structures. This software is freely available.
MIPS (Multiple, Iterative Primer Selector). The software MIPS outper-
forms HYDEN on the task of designing multiplex PCR experiments for
SNP genotyping. This software is freely available.
HYDEN (HighlY DEgeNerate primers) is a software useful for con-
structing primers and primer pairs with high degeneracy and yet high spec-
ificity. This software is freely available.
Use of degenerate primers can greatly reduce the specificity of the PCR
amplification, a problem that can be partly solved by using touchdown PCR.

Genetic code (IUPAC)

Code Amino acid Stands for
A A Adenine
C C Cytosine
G G Guanine
T T Thymine
U U Uracil
R A or G puRine
Y C or T (U) pYrimidine
M A or C aMino
K G or T (U) Keto
S C or G Strong (triple hydrogen bonds)
W A or T (U) Weak (double hydrogen bonds)
B C or G or T (U) not A
Continued
16 Rubén Álvarez-Fernández

D A or G or T (U) not C
H A or C or T (U) not G
V A or C or G not T (U)
N A or C or G or T (U) aNy nucleotide

1.16. Modified primers

Modifications can be positioned at the 30 - or 50 -end of the oligonucleotide,
or internally within the sugar-phosphate backbone or at the nucleobases.
These modified primers are used in applications such as in situ hybridization,
sequencing, fragment analysis, real-time PCR, fluorescent assays, chemilu-
minescent assays, microscopy, antisense experiments, and gene cloning and
discovery (e.g., degenerate primers). The modifications can also allow for
the oligonucleotides to be immobilized onto a surface, enabling spot-
detection (e.g., in microarrays, chemiluminiscent, and fluorescent assays).
These modifications include
• functionalization with amino, phosphate, or thiol groups
• fluorescent dyes
• hapten or enzyme coupling
• unnatural bases, for example, Br-dU, I-dU
• phosphothioates
• multiply-labeled oligonucleotides, for example, doubly labeled probes
• Locked Nucleic Acid (LNA™*)
The applications of each modification fall beyond the scope of this chapter,
but they can be found at major oligonucleotide supplier’s Web sites (e.g.,
Invitrogen, Sigma-Aldrich, IDT DNA, GeneLink, Exiqon, Thermo Elec-
tron, etc., in no particular order).

1.17. Recombineering
Recombineering stands for in vivo homologous recombination-mediated
genetic engineering. This technique relies on bacteriophage-based recom-
bination systems such as l Red, or RedET, which mediate recombination
between the host’s genome and a construct. Restriction-ligation techniques
* LNA nucleosides are a class of nucleic acid analogues in which the ribose ring is ‘locked’ by a methylene
bridge connecting the 20 -O atom and the 40 -C atom. This modification enhances the base pairing,
increasing the Tm and then the specificity, making them very useful in a variety of applications such
as Northern blotting, in situ hybridization, and real-time PCR, microarrays (see [Link]
[Link]/ for further information).
Explanatory Chapter: PCR Primer Design 17

are not used, thereby allowing for manipulation of longer constructs and
more versatility, including marker-free approaches (Karsten Tischer et al.,
2006). Recombineering with ssDNA oligonucleotides has been shown
to be very efficient (Costantino and Court, 2003); however, it can also
be performed with dsDNAs.
Primer design is key for efficient recombineering (Sawitzke et al., 2007.
See Recombineering: Highly Efficient in vivo genetic engineering using
single-strand oligos):
• The primer should correspond in sequence to the DNA strand that is
replicated discontinuously (the lagging-strand). Twenty-fold reductions
in efficiency are expected if the leading strand is chosen.
• It should be 70 bases long, and the base changes no closer than 10 bases
from an end. The longer the oligo, the more likely it is to have errors
introduced during synthesis. However, further purification is not
typically helpful.
• It should be designed to avoid the DNA-mismatch repair (MMR) sys-
tem (otherwise, the recombineering should be performed in a MMR-
deficient strain), following these guidelines:
• it should create a C/C mispair at the target base or 6 bases away from
it (Costantino and Court, 2003),
• or change 5 bases in a row,
• or change 4–5 wobble positions in a row, in addition to the
designed change.
For troubleshooting and the most up-to-date information on recombineering,
see [Link]

1.18. Primers for homologous recombination

Gene replacement can be performed in yeast by homologous recombination
using PCR products. Primer design is critical for this purpose, and some
guidelines are given below and those should be taken into account apart
from those at the beginning of this chapter (see the full protocol in Yeast -
Gene Replacement Using PCR Products).
• Homology to the gene to be replaced or interrupted: 50 nucleotides are
recommended and up to 100 nucleotide-long homology regions could
be needed. In some cases, as little as 40 nucleotides might work. If the
gene is to be replaced, the homology regions have to be upstream and
downstream of the coding sequence.
• Homology to the selectable cassette: 20 nucleotides are recommended.
18 Rubén Álvarez-Fernández

• Since the primers are > 70 nucleotides long, additional purification rather
than simply desalting might be needed (see Purification of DNA
Oligos by Denaturing Polyacrylamide Gel Electrophoresis (PAGE)).

1.19. Software tools list

Please note that no commercial interests have influenced the software choice
below, where an attempt has been made to balance the use of freely available
and commercial software. This review is not intended as an in-depth
comparison and marking of software tools, hence although these ones will
perform well for your primer design, better ones might be available in the
market for more specific applications.
AutoDimer (Vallone and Butler, 2004). [Link]
strbase/NIJ/[Link]
CODEHOP (Rose et al., 2003; Boyce et al., 2009). [Link]
[Link]/i-codehop-context/Welcome
HYDEN (Linhart and Shamir, 2002; 2005). [Link]
hyden/[Link]
JCVI Primer Designer (Li et al., 2008). [Link]
primerdesigner/
MAD-DPD (Najafabadi et al., 2008). [Link]
load/MAD_DPD08172007.zip
MIPS (Souvenir et al., 2003; 2007). [Link]
7Ezhang/projects/[Link]
NetPrimer. [Link]
OLIGO. [Link]
PAMPS (Najafabadi et al., 2008). [Link]
content/supplementary/[Link]
Primer3 (Rozen and Skaletsky, 2000). [Link]
Primer3Plus (Untergasser et al., 2007). [Link]
cgi-bin/primer3plus/[Link]
PrimerStation (Yamada et al., 2006) [Link]
Pythia (Mann at al., 2009). [Link]
ThermoBLAST™. [Link]
UNAFold (Mfoldþþ, Markham and Zuker, 2008). [Link]
[Link]/[Link] [Link]
Vector NTI®. [Link]
Online-Guides/LINNEA-Communities/Vector-NTI-Community/Vector-
[Link]
Visual OMP™. [Link]
Explanatory Chapter: PCR Primer Design 19

ACKNOWLEDGMENTS
The author thanks Dr. Sara Lopez-Gomollon for making a comprehensive review of
the manuscript.

REFERENCES
Referenced Literature
Boyce, R., Chilana, P., & Rose, T. M. (2009). iCODEHOP: A new interactive program for
designing COnsensus-DEgenerate Hybrid Oligonucleotide Primers from multiply
aligned protein sequences. Nucleic Acids Research 37(web server issue). [Link]
10.1093/nar/gkp379.
Chamberlain, J. S., Gibbs, R. A., Rainer, J. E., Nguyen, P. N., & Thomas, C. (1988). Dele-
tion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplifi-
cation. Nucleic Acids Research, 16, 11141–11156.
Costantino, N., & Court, D. L. (2003). Enhanced levels of l Red-mediated recombinants in
mismatch repair mutants. Proceedings of the National Academy of Sciences of the United States
of America, 100, 15748–15753. [Link]
Dieffenbach, C. W., Lowe, T. M., & Dveksler, G. S. (1993). General concepts for PCR
primer design. Genome Research, 3, S30–S37.
Karsten Tischer, B., von Einem, J., Kaufer, B., & Osterrieder, N. (2006). Two-step Red-
mediated recombination for versatile high-efficiency markerless DNA manipulation
in Escherichia coli. BioTechniques, 40, 191–197. [Link]
Li, K., Brownley, A., Stockwell, T. B., et al. (2008). Novel computational methods for
increasing PCR primer design effectiveness in directed sequencing. BMC Bioinformatics,
9, 191. [Link]
Linhart, C., & Shamir, R. (2002). The degenerate primer design problem. Bioinformatics, 18,
S172–S180.
Linhart, C., & Shamir, R. (2005). The degenerate primer design problem: Theory and appli-
cations. Journal of Computational Biology, 12, 431–456. [Link]
10.1089/cmb.2005.12.431.
Liu, Y.-G., & Whittier, R. F. (1995). Thermal asymmetric interlaced PCR: Automatable
amplification and sequencing of insert end fragments from PI and YAC clones for chro-
mosome walking. Genomics, 25, 674–681.
Mann, T., Humbert, R., Dorschner, M., Stamatoyannopoulos, J., & Noble, W. S. (2009).
A thermodynamic approach to PCR primer design. Nucleic Acids Research, 37(13), e97.
[Link]
Markham, N. R., & Zuker, M. (2008). UNAFold: Software for nucleic acid folding and
hybriziation. ch. 1,In J. M. Keith (Ed.), Methods in Molecular Biology: vol. 453. Bioinfor-
matics, Volume II. Structure, Function and Applications (pp. 3–31). Totowa, NJ: Humana
Press.
Mülhardt, C. (2007). The polymerase chain reaction. In Molecular Biology and Genomics
(pp. 65–94): Elsevier.
Najafabadi, H. S., Saberi, A., Torabi, N., & Chamankhah, M. (2008). MAD-DPD: Design-
ing highly degenerate primers with maximum amplification specificity. BioTechniques,
44, 519–526.
Najafabadi, H. S., Torabi, N., & Chamankhah, M. (2008). Designing multiple degenerate
primers via consecutive pairwise alignments. BMC Bioinformatics, 9, 55. [Link]
org/10.1186/1471-2105-9-55.
Rose, T. M., Henikoff, J. G., & Henikoff, S. (2003). CODEHOP (COnsensus-DEgenerate
Hybrid Oligonucleotide Primer) PCR primer design. Nucleic Acids Research, 31,
3763–3766. [Link]
20 Rubén Álvarez-Fernández

Rozen, S., & Skaletsky, H. J. (2000). Primer3 on the WWW for general users and for biol-
ogist programmers. In S. Krawetz & S. Misener (Eds.), Bioinformatics Methods and Protocols:
Methods in Molecular Biology (pp. 365–386). Totowa, NJ: Humana Press.
Rychlik, W. (1990). Optimization of the annealing temperature for DNA amplification
in vitro. Nucleic Acids Research, 18, 6409–6412.
Rychlik, W. (1993). Selection of primers for polymerase chain reaction. In B. A. White (Ed.),
Methods in Molecular Biology: vol. 15. PCR Protocols: Current Methods and Applications
(pp. 31–40).
SantaLucia, J., Jr. (2007). Physical principles and visual-OMP software for optimal PCR
design. In A. Yuryev (Ed.), Methods in Molecular Biology: vol. 402. PCR Primer Design
(pp. 245–267). Totowa, NJ: Humana Press.
SantaLucia, J., Jr. (1998). A unified view of polymer, dumbbell, and oligonucleotide DNA
nearest-neighbor thermodynamics. Proceedings of the National Academy of Sciences of the
United States of America, 95, 1460–1465.
Sawitzke, J. A., Thomason, L. C., Costantino, N., Bubunenko, M., Datta, S., & Court, D. L.
(2007). Recombineering: In vivo genetic engineering in E. coli, S. enterica, and beyond.
In K. T. Hughes & S. R. Maloy (Eds.), Methods in Enzymology: vol. 421. Advanced
Bacterial Genetics: Use of Transposons and Phage for Genomic Engineering (pp. 171–199).
San Diego, CA: Academic Press. 10.1016/S0076-6879(06)21015-2.
Souvenir, R., Buhler, J., Stormo, G., & Zhang, W. (2003). Selecting degenerate multiplex
PCR primers. Proceedings of the 3rd Workshop on Algorithms in Bioinformatics (WABI 2003)
(pp. 512–526).
Souvenir, R., Buhler, J., Stormo, G., & Zhang, W. (2007). PCR primer design. In A. Yuryev
(Ed.), pp: 402. Methods in Molecular Biology (pp. 245–267). Totowa, NJ: Humana Press.
Untergasser, A., Nijveen, H., Rao, X., Bisseling, T., Geurts, R., & Leunissen, J. A. M.
(2007). Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Research,
35, W71–W74. [Link]
Vallone, P. M., & Butler, J. M. (2004). AutoDimer: A screening tool for primer-dimer and
hairpin structures. BioTechniques, 37, 226–231.
Viguera, E., Canceill, D., & Ehrlich, S. D. (2001). In vitro replication slippage by DNA poly-
merases from thermophilic organisms. Journal of Molecular Biology, 312, 323–333. http://
[Link]/10.1006/jmbi.2001.4943.
Wong, M. L., & Medrano, J. F. (2005). Real-time PCR for mRNA quantitation.
BioTechniques, 39, 1–11.
Yamada, T., Soma, H., & Morishita, S. (2006). PrimerStation: A highly specific multiplex
genomic PCR primer design server for the human genome. Nucleic Acids Research
34(web server issue): (pp. W665–W669). [Link]
Zhou, M.-Y., & Gomez-Sanchez, C. E. (2000). Universal TA cloning. Current Issues in
Molecular Biology, 2, 1–7.

Related Literature
Primer Design, P. C. R. (2007). In A. Yuryev (Ed.), Methods in Molecular Biology: vol. 402.
Totowa, NJ: Humana Press.
[Link]

Referenced Protocols in Methods Navigator

General PCR
Explanatory Chapter: Troubleshooting PCR
Purification of DNA Oligos by Denaturing Polyacrylamide Gel Electrophoresis (PAGE)
Molecular Cloning
Restrictionless cloning
Explanatory Chapter: PCR Primer Design 21

Reverse-transcription PCR (RT-PCR)

Northern blotting
Explanatory Chapter: Nuclease Protection Assays
Explanatory Chapter: Quantitative PCR
Recombineering: Highly Efficient in vivo genetic engineering using single-strand oligos
Yeast - Gene Replacement Using PCR Products