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PCR Primer Design of Medicilon

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A primer is a short synthetic oligonucleotide which is used in many molecular techniques from PCR to DNA sequencing.  These primers are designed to have a sequence which is the reverse complement of a region of template or target DNA to which we wish the primer to anneal.

pcr primer design

Analysis of Primer Sequences

When designing primers for PCR, sequencing or mutagenesis it is often necessary to make predictions about these primers, for example melting temperature (Tm) and propensity to form dimers with itself or other primers in the reaction.  The following program will perform these calculations on any primer sequence or pair.

The programs will calculate both the Tm of the primers, as well as any undesireable pairings of primers.  When primers form hairpin loops or dimers less primer is available for the desired reaction.

Primer Design Considerations

One of the single most important factors in successful automated DNA sequencing is proper primer design.  It is important that a primer has the following characteristics:

A melting temperature (Tm) in the range of 50 C to 65 C

Absence of dimerization capability

Absence of significant hairpin formation (>3 bp)

Lack of secondary priming sites

Low to moderate specific binding at the 3′ end (avoid high GC content to prevent mispriming)

Primers designed according to these criteria will generally be from 18 to 30 bases in length and have %GC of 40 to 60. Try to avoid using primers with Tm’s above 65-70 C, especially on high GC templates, as this can lead to secondary priming artifacts and noisy sequences. We strongly recommend the use of computer software to design primers with these characteristics. Examples of such software are: LaserGene (DNAStar), Oligo (National Biosciences, Inc.), MacVector (Kodak/IBI) and the GCG suite.

If designing a primer based on existing sequencing data, choose a priming site that is greater than 50 nucleotides away from the position where new sequence is needed. Avoid designing primers using regions of poorer quality sequence, such as areas beyond single peak resolution of a chromatogram (typically 600-700 bases). Avoid primers where alternative priming sites are present with more than 90% identity to the primary site or that match at more than seven consecutive nucleotides at the 3′ end.

Finally, be aware that no set of guidelines will always accurately predict the success of a primer. Some primers may fail for no apparent reason, and primers that appear to be poor candidates may work well.

Designing Degenerate Oligonucleotides

A group of degenerate oligonucleotides contain related sequences with differences at specific locations.  These are used simultaneously in the hope that one of the sequences of the oligonucleotides will be perfectly complementary to a target DNA sequence.

One common use of degenerate oligonucleotides is when the amino acid sequence of a protein is known.  One can reverse translate this sequence to determine all of the possible nucleotide sequences that could encode that amino acid sequence.  A set of degenerate oligonucleotides would then be produced matching those DNA sequences.  The following link will take you to a program that will perform a reverse translation.

Also keep in mind that most oligonucleotide synthesis reactions are only 98% efficient.  This means that each time a base is added, only 98% of the oligos will receive the base.  This is not often critical with shorter oligos, but as length increases, so does the probability that a primer will be missing a base.  This is very important in mutagenesis or cloning reactions.  Purification by HPLC or PAGE is recommended in some cases.

PCR Primer Design

Polymerase Chain Reaction is widely held as one of the most important inventions of the 20th century in molecular biology. Small amounts of the genetic material can now be amplified to be able to a identify, manipulate DNA, detect infectious organisms, including the viruses that cause AIDS, hepatitis, tuberculosis, detect genetic variations, including mutations, in human genes and numerous other tasks.

PCR involves the following three steps: Denaturation, Annealing and Extension.


First, the genetic material is denatured, converting the double stranded DNA molecules to single strands. The primers are then annealed to the complementary regions of the single stranded molecules. In the third step, they are extended by the action of the DNA polymerase. All these steps are temperature sensitive and the common choice of temperatures is 94℃, 60℃ and 70℃ respectively. Good primer design is essential for successful reactions. The important design considerations described below are a key to specific amplification with high yield. The preferred values indicated are built into all our products by default.

1. Primer Length: It is generally accepted that the optimal length of PCR primers is 18-22bp. This length is long enough for adequate specificity, and short enough for primers to bind easily to the template at the annealing temperature.

2. Melting Temperature: Melting Temperature (Tm) by definition is the temperature at which one half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability. Primers with melting temperatures in the range of 52-58℃ generally produce the best results. Primers with melting temperatures above 65℃ have a tendency for secondary annealing. The GC content of the sequence gives a fair indication of the Tm. All our products calculate it using the nearest neighbor thermodynamic theory, accepted as a much superior method for estimating it, which is considered the most recent and best available.

3. Primer Annealing Temperature: The primer melting temperature is the estimate of the DNA-DNA hybrid stability and critical in determining the annealing temperature. Too high Ta will produce insufficient primer-template hybridization resulting in low PCR product yield. Too low Ta may possibly lead to non-specific products caused by a high number of base pair mismatches,. Mismatch tolerance is found to have the strongest influence on PCR specificity.

4. GC Content: The GC content (the number of G’s and C’s in the primer as a percentage of the total bases) of primer should be 40-60%.

5. GC Clamp: The presence of G or C bases within the last five bases from the 3′ end of primers (GC clamp) helps promote specific binding at the 3′ end due to the stronger bonding of G and C bases. More than 3 G’s or C’s should be avoided in the last 5 bases at the 3′ end of the primer.

6. Secondary Structures: Presence of the secondary structures produced by intermolecular or intramolecular interactions can lead to poor or no yield of the product. They adversely affect primer template annealing and thus the amplification. They greatly reduce the availability of primers to the reaction.

7. Repeats: A repeat is a di-nucleotide occurring many times consecutively and should be avoided because they can misprime. For example: ATATATAT. A maximum number of di-nucleotide repeats acceptable is 4 di-nucleotides.

8. Runs: Primers with long runs of a single base should generally be avoided as they can misprime. For example, AGCGGGGGATGGGG has runs of base ‘G’ of value 5 and 4. A maximum number of runs accepted is 4bp.

9. 3′ End Stability: It is the maximum ΔG value of the five bases from the 3′ end. An unstable 3′ end (less negative ΔG) will result in less false priming.

10. Avoid Template Secondary Structure: A single stranded Nucleic acid sequences is highly unstable and fold into conformations (secondary structures). The stability of these template secondary structures depends largely on their free energy and melting temperatures(Tm). Consideration of template secondary structures is important in designing primers, especially in qPCR. If primers are designed on a secondary structures which is stable even above the annealing temperatures, the primers are unable to bind to the template and the yield of PCR product is significantly affected. Hence, it is important to design primers in the regions of the templates that do not form stable secondary structures during the PCR reaction. Our products determine the secondary structures of the template using the M fold algorithm and design primers avoiding them.

11. Avoid Cross Homology: To improve specificity of the primers it is necessary to avoid regions of homology. Primers designed for a sequence must not amplify other genes in the mixture. Commonly, primers are designed and then BLASTed to test the specificity. Our products offer a better alternative. You can avoid regions of cross homology while designing primers. You can BLAST the templates against the appropriate non-redundant database and the software will interpret the results. It will identify regions significant cross homologies in each template and avoid them during primer search.

Parameters for Primer Pair Design

1. Amplicon Length : The amplicon length is dictated by the experimental goals. For qPCR, the target length is closer to 100bp and for standard PCR, it is near 500 bp. If you know the positions of each primer with respect to the template, the product is calculated as: Product length = (Position of antisense primer-Position of sense primer) +1.

2. Product Position : Primer can be located near the 5′ end, the 3′ end or any where within specified length. Generally, the sequence close to the 3′ end is known with greater confidence and hence preferred most frequently.

3. Tm of Product : Melting Temperature (Tm) is the temperature at which one half of the DNA duplex will dissociate and become single stranded. The stability of the primer-template DNA duplex can be measured by the melting temperature(Tm).

4. Optimum Annealing Temperature (Ta Opt): The formula of Rychlik is most respected. Our products use this formula to calculate it and thousands of our customers have reported good results using it for the annealing step of the PCR cycle.It usually results in good PCR product yield with minimum false product production.

5. Primer Pair Tm Mismatch Calculation : The two primers of a primer pair should have closely matched melting temperatures for maximizing PCR product yield. The difference of 5℃ or more can lead no amplification.

Related Articles:

what is PCR (Polymerase Chain Reaction)?

PCR Primer Design of Medicilon

PCR Primer Analysis

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