Custom PCR Primer Synthesis

BOC Sciences offers flexible primer design and custom synthesis options to fit your experimental needs. We monitor the synthesis of each primer and deliver them to each customer for direct application in a variety of downstream experiments.

What is PCR Primer?

PCR primers are synthetic DNA oligonucleotides containing 15-30 bases that bind to the flanking sequences of the target region in the template DNA (through sequence complementation). During the PCR reaction, DNA polymerase extends the primer from the 3' end. Therefore, the primer binding site must be unique to the vicinity of the target and have minimal sequence homology to the rest of the starting DNA to ensure specific amplification of the target fragment.

Fig 1. Schematic of primer design. (Ogren et al., 2019)

PCR Primer Synthesis Service

PCR primer synthesis and design can usually be found in primer libraries, or custom synthesized according to the needs of low-cost, easy to operate, etc. PCR primer synthesis can be used for sequencing, whole gene synthesis, subcloning, point mutation, vector construction, RNA interference and recombination, and other experiments.

• PCR Primer Design

  
  Primer design requires consideration of the primer length, GC content, complementarity, site selection and other factors, and the use of primer design software or online tools to assist the design.

• Multiplex PCR Primer Design

  
  Multiplex PCR primer is a PCR reaction that uses a template and several pairs of primers to amplify multiple target fragments in one PCR reaction. This technique is very useful not only to increase the yield of PCR but also to increase the utilization of the DNA sample. Multiplex PCR primer requires that all primer pairs amplify their respective specific sequences under the same conditions. One reason most multiplex PCR reactions are limited to amplifying 5-10 target fragments is that the addition of another primer pair to the reaction results in some loss of flexibility. Increasing the number of primers also increases the probability of primer dimerization and non-specific amplification. Therefore, performing multiplex PCR requires careful planning and several attempts to optimize the reaction conditions.

• Real-Time qPCR Primer Design

  
  In the process of real-time qPCR primer synthesis, primer design is a key factor for the success of the experiment, which largely determines the sensitivity and accuracy of the detection, generally, real-time qPCR primer design needs to follow the following principles.
  Generally, the primer length is between 17-25 bases, and the difference between the upstream and downstream primers should not be too big. Primer length is the most important factor in determining the annealing temperature (Tm value). Generally speaking, each additional nucleotide increases the specificity of the primer by 4 times, but due to entropy, the longer the primer, the lower the rate at which it will anneal to the target DNA to form a stable double-stranded template for DNA polymerase binding.

• PCR Primer Synthesis

  
  Currently, the solid-phase phosphoramidite method is used for primer synthesis. This method is characterized by high efficiency, rapid coupling and relatively stable starting reactants. Mainly, the DNA is immobilized on the solid-phase carrier to complete the synthesis of the DNA strand, from the 3' end of the primer to be synthesized to the 5' end of the synthesis of the extension of the adjacent nucleotides through the 3'→5' phosphodiester bond connection.

• Purification Method of Primers

Advantages of BOC Sciences' PCR Primer Synthesis

• Advanced Equipment - With advanced synthesizer to make primer synthesis faster, safer and more stable.
• Rapid Delivery - Multiple primers within 30 bases can be synthesized in a short time.
• High-quality Products - Multiple purification methods and multiple quality testing methods (HPLC, MS) fully guarantee the quality of our shipments.

Frequently Asked Questions (FAQ)

What factors determine optimal PCR primer design for specific applications?

Optimal primer design considers multiple parameters including length (18-25 bases), GC content (40-60%), melting temperature compatibility, secondary structure avoidance, and 3'-end stability to ensure specific amplification efficiency.

How are multiplex PCR primers designed to avoid primer-dimer formation?

Multiplex primer design employs sophisticated algorithms to minimize complementary regions between different primers, optimize annealing temperature uniformity, and strategically position primer binding sites to prevent cross-hybridization.

What purification methods ensure high-quality primer synthesis?

Multiple purification options including PAGE, HPLC, and ePAGE purification effectively remove truncated sequences and synthesis byproducts, with selection based on application requirements and purity specifications.

How do real-time qPCR primers differ from conventional PCR primers?

qPCR primers require enhanced specificity with minimal secondary structure, optimized amplicon length (typically 75-150 bp), and validation to ensure efficient amplification with minimal primer-dimer interference in fluorescence-based detection.

Reference

1. Ogren A, et al. Snord94 expression level alters methylation at C62 in snRNA U6[J]. Plos one, 2019, 14(12): e0226035.