Oligonucleotide Design and Ordering Tips

Custom oligos are an important tool that researchers can use in a wide variety of applications. Here are important design and ordering tips to get you to your experiments faster!

Ideally, the GC content of an oligonucleotide primer should be between 40% and 60% with the 3’ end of the DNA sequence ending in G or C to promote binding (known as a GC clamp). Since G and C bases have stronger hydrogen bonding than A and T, incorporating them at the end of the sequence can help increase stability of the primer at the site of elongation. Be mindful, however, not to have too many repeating G or C bases as this can enhance primer-dimer formation.

The melting temperature (T_m) of the oligonucleotide primers should be between 65°C and 75°C, and within 5°C of each other. Because the T_m is dependent on the length of the sequence, it’s important to keep primers minimal in length, if possible. The individual bases of the sequence also impact the T_m, with G and C bases resulting in higher melting temperatures than A and T bases. If the T_m of your primer is very low, try to find a sequence with more GC content, or slightly extend the length of the primer.

Specificity of the oligonucleotide primer is usually dependent on the sequence composition, length, and annealing temperature. An optimal length for primers is 18–30 bases, though the right length is dependent on a number of factors. Shorter primers bind more efficiently to the target due to a lower relative T_m. However, short sequences may also suffer from lower specificity so a balance must be struck.

A common practice with primers used for cloning is to add restriction enzyme (RE) recognition sites flanking the insert sequence. This is done by including the appropriate RE cleavage site on the 5’ end of the primers used to amplify the insert sequence. It is important to include an additional 3–4 nucleotides to the 5’ end of the RE sites to help ensure efficient cleavage when preparing the amplicon for cloning.

The primary sequence of the oligo influences annealing to the intended template. For example, if the sequence allows for a hairpin secondary structure, this can render the primer unavailable for binding with the template. Similarly, if the sequence favors complementary binding to either another copy of the same primer sequence or the sequence of the paired primer (forward and reverse primer), this can lead to amplification of a very short product rather than the desired amplicon. In addition, runs of four or more of a single base or two base repeats (i.e., ACCCC, ATATATAT) can cause mis-priming on the template DNA.

Mutagenesis is a technique for introducing small, specific mutations into the sequence during amplification of a plasmid. This is done by incorporating mismatched bases in the middle of complementary primers, leaving flanking sequence on either end to bind to the template. Refer to your mutagenesis kit instructions for detailed guidance on length and design of the primers.

TOPO cloning allows for efficient direct cloning of PCR products. Primers used for this method should not include a phosphate modification to ensure functionality with TOPO cloning kits.

With multiple parameters to consider when designing a DNA primer, it is helpful to have tools for assessing potential primer sequences. Thermo Fisher Scientific offers several tools to help simplify this process.

• OligoPerfect Designer—A comprehensive design tool with configurable parameters. The user can choose how many candidate primer pairs they would like in the results to further evaluate. The results include a visual layout of the candidate primers in relation to the template sequence, a sequence viewer and critical features of each candidate including T_m, % GC, and calculated hairpin melting temperature.
• T_m Calculator—Calculate the T_m for your primers specific to the polymerase you are using and the primer concentration in the reaction mix.
• Multiple Primer Analyzer—Check candidate sequences for potential primer dimers.

When ordering your oligos, it’s important to know the difference between starting scale and final yield. Every processing step (i.e., base addition, purification, desalting) and modification addition is a chemical reaction that can diminish the yield of your oligo. When using the custom standard oligo ordering tool, the minimum guaranteed amount based on sequence design and oligo options, will be displayed. If the amount will not work for your experiments, consider using a larger synthesis scale or modifying your selected options/modifications.

While it’s true that purification options remove truncated sequences from the oligo sample to increase sample purity, this level of purification is not always necessary. The level of purity you need will depend on the application that you are using the oligo for, the length of the oligo, the desired final yield, and the modifications added to the oligo. For PCR or sequencing applications that don’t require ≥85% purity and use oligos that do not contain modifications, standard processing with ready-to-use-desalting options will often be the preferred choice. For applications like cloning, next-generation sequencing (NGS), or mutagenesis, or for oligos that contain modifications, high purity is critical and will require additional purification options using cartridge, HPLC, or PAGE methods.

Designing a custom oligo for end-point PCR, CE sequencing, or cloning is easy using our dynamic OligoPerfect Designer. The Primer3-based tool offers several primers or primer pair options for you to choose from. After selection, primers can be reviewed for their length, GC content, T_m, and price before adding to your online cart for checkout.*

Using custom oligos in your research can get you to your results faster. We have optimized the design and ordering process to make your experience easy! For more information on custom oligo services, please see the links below.

* Pricing and add to cart feature may vary by geographic region. For questions, please contact your local office or distributor.