Designing PCR Primers Painlessly (2024)

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).

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 nucleotides, 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 primers that were then used to amplify and clone several genes 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 designing 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, P_BAD, 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 regulatory 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 directionality 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.

SUPPLEMENTAL MATERIALS

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

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