Primers can trigger DNA polymerase-mediated DNA synthesis by providing the 3'-end of hydroxyl group when complementarily hybridizing to the target DNA. For successful PCR amplification, the synthesis of primers is also one of the key factors as they are primarily responsible for determining PCR specificity. On the basis of early rational design, the synthesis work needs further consideration.
Currently, primer synthesis can be primarily achieved by using special nucleic acid synthesizing machines, which can synthesize primers with maximum lengths of about 70 nucleotides. The chemical mechanism relies on a series of non-enzymatic, organic chemistry reactions. The most common methods are based on β-cyanoethyl phosphoamidite and the H-phosphonate cycle. Figure 1 shows a general scheme of the β-cyanoethyl phosphoramidite method.
Figure 1. Oligonucleotide synthesis using the β-cyanoethyl phosphoramidite method. (Pelt-Verkuil E., et al. 2008)
- Before reactions, all the reagents will be stored separately in different compartments of the DNA synthesiser. Notably, the reactive sites of these reagents are blocked so that chemical reactions can only occur when acid or alkaline is used to remove these blocking groups.
- Detritylation: The first step is to remove the 5' -end blocking group (dimethoxytrityl, DMT) by dichloro-acetic acid treatment, which will result in an exposed hydroxyl group of an immobilsed nucleotide.
- Activation: Tetrazole will be used to active the 3'-end of another nucleotide.
- Coupling: This activated nucleotide will be coupled to the 5'-end of the nucleotide obtained in the first step via a condensation reaction.
- Oxidation: Iodide can be used to oxidize the phosphite ester into a phosphate ester.
- Capping: In order to avoid generated unwanted truncated oligonucleotides, all of the 5'-end hydroxyl groups should be capped as a preventive step.
Creative Biogene can provide one-stop custom PCR primer design and synthesis service with high quality and high efficiency to keep up with the demands for various primers. The following information includes all the options that you can select.
- Various scales (from 0.05 to 5 µM per oligonucleotide) can be synthesized.
- Both forward and reverse primers are available.
- Different types of chemical schemes are available, such as phosphoramidite and phosphothioate methods.
- The purity of the final product can be customized according to your needs.
- Different kinds of labelling (such as biotin, FAM, FITC, etc.), base moieties (such as inosine, uracil), random sequences, degenerate nucleotide positions can be customized.
- Primers can be packed and delivered in solution or as a lyophilized powder.
- Pelt-Verkuil E., Belkum A., Hays J P. Principles and technical aspects of PCR amplification Volume 7504 || PCR primers. 10.1007/978-1-4020-6241-4. Chapter 5, 2008, 63-90.