Primer Design Services

Primers refer to short single-stranded DNA or RNA sequences complementary to target DNA sequences, which are responsible for initiating the process of DNA replication. Normally, RNA primers work for cellular DNA replication exclusively. Instead, DNA primers play an essential role in PCR amplification of DNA in vitro as they are easily prepared by DNA Primase enzyme. So far, different DNA primers have been designed and improved to meet corresponding requirements.  

Simple guidance of primer design

As one of the most crucial components for PCR assays, primer matters for the assays' specificity, sensitivity, and robustness. And quite a few reports have emphasized that there are some parameters that need to be taken into account when processing a primer design.

  • Length
  • In most PCR applications, 18-30 bases in length could be optimal. Longer or shorter primers may cause nonspecific PCR amplification or containing some undesired secondary structures, respectively.

  • Melting temperature
  • The melting temperature (Tm) of a primer normally depends on the length, sequence composition (G/C can lead to higher Tm than A/T), and concentration, whose optimum range is 55 - 60°C. Notably, the Tm of primer pair should be as similar as possible and their Tm mismatch should be less than 2°C - 3°C.

  • GC clamp
  • In order to enhance the priming efficiency, "GC clamp" can be introduced into the primer sequences by inserting a G or C residue at the 3′-end. As we all know, GC rather than TA can be bound with stronger hydrogen bond.

  • GC content
  • Balancing of the distribution of GC and TA is another requirement in primer design. And the GC content of primers is generally 40-60%, preferably 45-55%. If the target DNA is a GC-rich sequence, a higher Tm of primer will be needed as the separation temperature of the two strands are always higher and their PCR amplicons may re-anneal faster at low temperature.

  • Secondary structure
  • Notably, intra- or inter-primer homologies as well as too many repeating G or C bases in primer sequence could result in self- or primer-dimer structures. One of the simple ways is to design primers containing 50% G + C in absence of one of the four bases.  

Our services

Our company can provide multiple primer design services. Detailed information is shown below:

  • Primers suitable for different applications can be designed, such as multiplex PCR primer, nested PCR, cDNA/gDNA selective primer design and cross-species primer design, etc.
  • Primer design can also be achieved with kinds of molecular biological software, including Oligo 6、Premier Premier、Primer 3、Vector NTI Suit、Dnasis、Omiga、Dnastar, etc.
  • A set of completed and perfect design systems will be provided, including initial design and screening, secondary screening (focusing on primer pair with high specificity and high efficiency), modifications on primers (attaching fluorescein, digoxin, biotin, etc.) and final assessment through PCR amplification (the specificity, efficiency, yield, etc.).

References:

  1. Li K, Brownley A. Primer design for RT-PCR. Methods In Molecular Biology, 2010, 630: 271-299.
  2. Dieffenbach C W, Lowe T M, Dveksler G S. General concepts for PCR primer design. PCR Methods and Applications, 1993, 3(3): S30-S37.
  3. Nybo K. DNA and general PCR methods: PCR primer design. Biotechniques, 2009, 46(7): 505-507.
  4. Thornton B, Basu C. Rapid and simple method of qPCR primer design. Methods In Molecular Biology, 2015, 1275: 173-179.
  5. Kumar A, Chordia N. In silico PCR primer designing and validation. Methods In Molecular Biology, 2015, 1275: 143-151.
* It should be noted that our service is only used for research, not for clinical use.

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