Advances in Primer and Probe Synthesis and Purification Technologies and Selection of Suitable Options

Solid-phase synthesis and common modification capabilities

Primer and probe synthesis is mostly carried out by the solid-phase phosphoramidite method, in which sequence extension is achieved through stepwise coupling of nucleotide monomers. This technology can support multiple base types such as DNA, RNA, locked nucleic acid (LNA), and phosphorothioate modifications. For probes, 5' fluorescent labeling, 3' quencher labeling, and internal modifications are commonly used. During synthesis, fluorescent groups can be introduced through phosphoramidite monomers or post-synthetic labeling. The choice of modifying groups needs to take into account the probe's enzymatic cleavage requirements, fluorescence signal intensity, and hybridization stability. It is generally recommended to communicate with the synthesis service provider at the design stage regarding available modification types and labeling positions to avoid signal reduction caused by inappropriate modifications.

Comparison and selection of purification methods

The crude product after synthesis often contains truncated sequences, salt ions, and organic byproducts, and purification is required to improve product purity. Common purification methods include desalting, reverse-phase high-performance liquid chromatography (RP-HPLC), polyacrylamide gel electrophoresis (PAGE), and ion-exchange purification. Desalting only removes salts and is suitable for ordinary PCR primers; PAGE has a good separation effect on long-chain products and truncated sequences and is suitable for long probes or high-purity requirements; HPLC has higher purification efficiency for fluorescently labeled probes and can remove unlabeled free dyes. The specific choice should be based on product length, modification complexity, and the sensitivity requirements of the experiment.

Matching synthesis scale with application

The synthesis scale of primers and probes is generally at the nanomole to micromole level. Laboratory research usually chooses small-scale synthesis, while in vitro diagnostic reagent production may require larger-scale custom synthesis that meets quality system requirements. Large-scale synthesis requires attention to intra-batch uniformity, purification recovery rate, and cost balance. For multiplex detection, it may be necessary to use a combination of probes labeled with different fluorescent groups, and in this case the degree of emission spectral overlap among the probes should be confirmed. In addition, regarding storage form, lyophilized powder is more conducive to long-term storage than liquid, and after reconstitution it should be aliquoted for storage to reduce repeated freeze-thaw cycles.

Rational selection of primer and probe synthesis and purification routes helps control R&D or production costs while ensuring detection performance. It is recommended to conduct small-sample validation based on the specific experimental system before scaling up.