Tips & Tools
JOURNAL OF MICROBIOLOGY & BIOLOGY EDUCATION, May 2014, p. 28-29
DOI: http://dx.doi.org/10.1128/jmbe.v15i1.634
Designing PCR Primers Painlessly †
Morgan Feeney, Kevin Murphy, and Jane Lopilato*
Biology Department, Simmons College, Boston, MA 02115
INTRODUCTION
Students often find designing primers for amplifying
genes by PCR a painful and frustrating experience. We
have devised and tested a simple computer- and paper-
based method that minimizes the confusion and produces
usable primers that can be used in the laboratory or as an
exercise in the classroom to introduce bioinformatics (2).
The students block off the DNA sequences at the beginning
of their gene for the forward primer and at the end of the
gene for their reverse primer and add a restriction enzyme
site along with a few extra nucleotides to each primer. They
then determine the melting temperatures of their primers
and those closest in temperature can be ordered and used
for amplifying and cloning genes.
PROCEDURE
There are no safety issues involved in designing primers.
Basic laboratory safety procedures are needed for cloning
and bacterial transformation.
The only materials required are a paper copy of a
prokaryotic gene to be amplified, a map of the vector into
which it will be cloned, and access to the Internet. We have
used genes from Escherichia coli, most recently, slyA. Students
begin by examining the multiple cloning sites of the vector
and choose two restriction enzyme sites, for example EcoR1
and HindIII, to use in cloning their gene. The paper copy of
their gene includes upstream and downstream sequences
in addition to the coding sequence downloaded from the
xBASE site (http://www.xbase.ac.uk/). The advantage of
using xBASE is that the upper strand of the DNA sequence
is the coding strand with the amino acids indicated, while
the lower strand is the template strand written in lower
case (Appendix 1). This format makes it easy for students
to see what a prokaryotic gene looks like and immediately
find the start and stop codons to better understand gene
structure (3).
*Corresponding author. Mailing address: Biology Department,
Simmons College, 300 The Fenway, Boston, MA 02115. Phone: 617-
521-2661. Fax: 617-521-3086. E-mail: jane.lopilato@simmons.edu.
†Supplemental materials available at http://jmbe.asm.org
©2014 Author(s). Published by the American Society for Microbiology. This is an Open Access article distributed under the terms of the a Creative Commons Attribution – Noncommercial –
Share Alike 3.0 Unported License (http://creativecommons.org/licenses/by-nc-sa/3.0/), which permits unrestricted non-commercial use and distribution, provided the original work is properly cited.
28 Journal of Microbiology & Biology Education
The students work in pairs and open EcoCyc (http://
ecocyc.org/), New England Biolabs (NEB) Cutter (http://
tools.neb.com/NEBcutter2/index.php), and Integrated
DNA Technologies OligoAnalyzer 3.1 (http://www.idtdna.
com/analyzer/applications/oligoanalyzer/) in separate tabs
(Appendix 2). From EcoCyc, they copy the single coding
strand of their gene and paste it into NEB Cutter. Then
the sequence of their gene is examined in the cutter for all
enzymes. Going to the “0 cutting” information, they can
see if either of the enzymes chosen, EcoR1 and HindIII in
the example above, has restriction enzyme sites and cuts
within their gene. If their gene lacks both sites, then they
start designing their primers.
For the forward primer, a few nucleotides, usually Gs
or Cs, are added onto the 5’ end of the restriction site
of first enzyme, here EcoR1. These extra bases ensure
efficient digestion of the PCR products by the restriction
enzymes (NEB). Following the restriction enzyme site,
the sequence of the gene beginning with the start codon
(ATG or sometimes TTG) is added. The students attach
the sequence from the “top” or coding strand of their
gene and simply write the nucleotides from left to right
(usually 12–18 bases).
The reverse primer is a little more complicated: first
the students write the extra nucleotides and then the
restriction site, HindIII, from 5’ to 3’. Following that, they
add the DNA sequence of the “bottom” or template strand
starting with the stop codon and proceeding right to left
into their gene, which is also 5’ to 3’ (Appendix 1). The
students add approximately 12 to 18 nucleotides identical
to the sequence of their gene and try to end with a G or
a C when possible.
Once the students have the initial sequence of their
forward and reverse primers including the 5’ extra nucleo-
tides, the restriction sites, and the gene sequence, they
enter their DNA sequences into IDT’s OligoAnalyzer to
calculate the melting temperature of each primer. The
students are asked to design a pair of primers that differ by
no more than one degree. They quickly learn to adjust the
melting temperatures by adding or removing nucleotides
and see the effect of As and Ts versus Gs and Cs, while
still retaining the exact sequence of their gene.
We have used this method successfully to design prim-
ers that were then used to amplify and clone several genes
Volume 15, Number 1
 FEENEY et al.: DESIGNING PCR PRIMERS PAINLESSLY
into a variety of vectors (Appendix 3). Putting the primer
design in the context of an actual experiment enabled the
students to see why primers were required and could be
used in other exercises (5). In a typical semester, students
complete an entire cloning exercise, starting from design-
ing primers and purifying vector DNA to completing and
verifying the cloning. This past semester, the Molecular
Biology students amplified and cloned the slyA gene into the
pBAD24 vector (1), which puts the gene under the control
of the araBAD promoter, PBAD, and makes slyA expression
arabinose inducible. Using a protein expression vector
such as pQE-31 (QIAGEN), which adds a His tag, makes
the students think about reading frames: the sequence of
their gene must be in the same frame as the His tag. With
a lacZ vector, pRS415, for generating transcriptional fusions
(4), students had to examine the upstream region of their
gene to try to locate the promoter and necessary regula-
tory sequences using information from EcoCyc.
CONCLUSION
These tips can be used as a primer design exercise
or to generate primers for PCR as we have done in our
Molecular Biology lab sections. Students quickly realize
that their primers must be at the beginning and the end
of the gene to amplify the entire gene and that direc-
tionality is important: both primers cannot anneal to the
same strand of DNA. Students can take the next step
and write some of the DNA sequence each primer will
generate in an actual PCR reaction to better understand
DNA synthesis. The result is much less frustration and
better primer design.
Volume 15, Number 1 Journal of Microbiology & Biology Education
SUPPLEMENTAL MATERIALS
Appendix 1: Figure 1. Design and sequence of the slyA
primers along with the DNA sequence
from xBASE
Appendix 2: Screen shots of the websites used in the
primer design
Appendix 3: Sources for vectors, primers, enzymes,
and kits for DNA purification and isolation
ACKNOWLEDGMENTS
Many thanks to Jamie Traynor of Simmons College
Technology for the images in the appendices. The authors
declare that there are no conflicts of interest.
REFERENCES
1. Guzman, L .-M., D. Belin, M. J. Carson, and J.
Beckwith. 1995. Tight regulation, modulation, and high-
level expression by vectors containing the arabinose PBAD
promoter. J. Bacteriol. 177:4121–4130.
2. Klein, J. R., and T. Gulsvig. 2012. Using bioinformatics
to develop and test hypotheses: E. coli-specific virulence
determinants. J. Microbiol. Biol. Educ. 13:161–169.
3. May, B. J. 2013. Engaging students in a bioinformatics activity
to introduce gene structure and function. J. Microbiol. Biol.
Educ. 14:107–109.
4. Simons, R. W., F. Houman, and N. Kleckner. 1987.
Improved single and multicopy lac-based cloning vectors for
protein and operon fusions. Gene 53:85–96.
5. Wright, L. K., and D. L. Newman. 2013. Using PCR to
target misconceptions about gene expression. J. Microbiol.
Biol. Educ. 14:93–100.
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