1. How are primers synthesized?
Currently, primer synthesis is fundamentally performed using the solid-phase phosphoramidite triester method. This method features high efficiency, rapid coupling, and relatively stable starting reactants. It primarily involves fixing DNA onto a solid support to accomplish the synthesis of the DNA chain. The synthesis extends from the 3' end to the 5' end of the target primer, with adjacent nucleotides linked via 3'5' phosphodiester bonds.
The specific steps for primer synthesis using the solid-phase phosphoramidite triester method are as follows:
- Trichloroacetic acid (TCA) is used to remove the DMT protecting group from the 5'-hydroxyl group of the solid support, yielding a free 5'-hydroxyl group.
- The phosphoramidite-protected nucleotide monomer is mixed with the activator tetrazole to generate an activated phosphoramidite intermediate, which then undergoes a condensation reaction with the free 5'-hydroxyl group.
- Since a 100% condensation rate for the 5'-hydroxyl groups cannot be guaranteed, a tiny fraction of 5'-hydroxyl groups (less than 2%) may fail to react. These can be capped and terminated using acetic anhydride and 1-methylimidazole reagents. These truncated short fragments can be removed during subsequent purification.
- Under the action of an oxidizing agent, the phosphite form is converted into a more stable phosphotriester, rendering the DNA phosphate backbone more stable.
Through the four steps above, one deoxynucleotide is linked to the solid support. These steps are repeated until all required bases for synthesis are connected. The synthesis efficiency can be preliminarily monitored and evaluated during the process by observing the color of the cleaved DMT protecting groups.
2. What impurities are contained in the crude DNA synthesis product?
Mainly a small amount of failure sequences (truncated fragments) generated during the synthesis process.
3. What are the primer purification methods?
Currently, commonly used primer purification methods include: C18 desalting, RPC purification, ePAGE purification, PAGE purification, and HPLC purification.
Table 1. Introduction to major primer purification methods
| Purification Method | Detailed Description |
|---|---|
| RPC Purification | RPC purification isolates primers using a Reverse Phase Cartridge, sharing the same purification principle as reverse-phase HPLC. Compared to reverse-phase HPLC, RPC is an efficient and more economical purification method. The reverse-phase cartridge typically contains a hydrophobic matrix such as C18 silica gel, which binds DNA effectively, allowing cleaved protecting groups and short primer fragments to be easily washed off the reverse-phase column with water. RPC-purified primers can be applied in DNA sequencing, PCR, gene synthesis, etc. |
| ePAGE Purification | ePAGE utilizes specific adsorption of full-length sequences to remove short sequences and impurities from the product. Assisted by automated batch loading and purification equipment, it completes DNA purification more efficiently. Its purity meets the needs of most molecular biology experiments, such as qPCR, multiplex PCR, site-directed mutagenesis, RNA interference (gene constructs), plasmid DNA sequencing, whole gene synthesis, PCR cloning, etc. Featuring automation, short turnaround times, and high throughput, ePAGE delivers products of comparable purity and quality to PAGE at lower prices and shorter delivery cycles. |
| PAGE Purification | PAGE purification utilizes denaturing polyacrylamide gel electrophoresis to separate primer DNA, followed by recovering the target DNA from the gel. PAGE purification is a highly effective method for DNA purification, yielding a post-purification DNA purity of greater than 90%. It is particularly effective for the purification of long-chain Oligo DNA (greater than 50mer). |
| HPLC Purification | HPLC purification applies High-Performance Liquid Chromatography principles to purify primer DNA. This method achieves high purity and sensitivity when used for separation, purification, or analysis. In primer DNA analysis and purification, Ion-Exchange HPLC and Reverse-Phase HPLC are commonly used. Reverse-Phase HPLC offers a purity greater than 90%, while Ion-Exchange HPLC achieves a purity greater than 95% and effectively removes N-1 failure fragments. HPLC purification is primarily used for short-chain and modified primers. The drawbacks of this method are its high cost and lower efficiency in mass production. |
4. How to choose the primer purification method?
GenScript offers four purification methods: RPC, ePAGE purification, PAGE, and HPLC. The selection primarily depends on the primer length and the purity requirements of the application. Table 2 summarizes the applicable scope of each purification method from the perspective of primer length. You can choose the appropriate primer purification method based on your experimental needs. For further guidance, please contact GenScript Technical Support.
Table 2. Applicable scope and recommendations for major primer purification methods
| Purification Method | Subtype | <15mer | 1540mer | 4159mer | 60120mer | Recommendation | Applicable Scope |
|---|---|---|---|---|---|---|---|
| RPC | - | N/A | Recommended | Applicable | N/A | Fast and high-throughput, commonly used for PCR and sequencing primers, etc. | Commonly used for PCR amplification, whole gene synthesis, and DNA sequencing, etc. |
| ePAGE | - | N/A | Recommended | Applicable | N/A | Purity satisfies the needs of most molecular biology experiments. | Commonly used for plasmid DNA sequencing, whole gene synthesis, site-directed mutagenesis (circular mutation), and PCR cloning, etc. |
| PAGE | - | N/A | Applicable | Applicable | Recommended | RPC or ePAGE purification is sufficient for routine molecular biology experimental primers; however, PAGE purification is strongly recommended for long-chain primers (>=60mer). | Unmodified oligonucleotides above 50mer, used for site-directed mutagenesis, cloning, protein-binding gel mobility shift assay (EMSA), therapeutic, and diagnostic purposes. |
| HPLC | Ion-Exchange | Recommended | Applicable | Applicable | N/A | Particularly effective for purifying short-chain primers (<15mer), but this method features lower throughput and higher costs. If the experiment requires exceptionally high purity, a dual refinement of HPLC and PAGE is recommended. | Unmodified oligonucleotides under 50mer, used for site-directed mutagenesis, cloning, protein-binding gel mobility shift assay, and therapeutic purposes; Modified primers with hydrophobic groups; Commercial diagnostic primers or probes (80%90% purity). |
| HPLC | Reverse-Phase | Recommended | Applicable | N/A | N/A | Particularly effective for purifying short-chain primers (<15mer), but this method features lower throughput and higher costs. If the experiment requires exceptionally high purity, a dual refinement of HPLC and PAGE is recommended. | Unmodified oligonucleotides under 50mer, used for site-directed mutagenesis, cloning, protein-binding gel mobility shift assay, and therapeutic purposes; Modified primers with hydrophobic groups; Commercial diagnostic primers or probes (80%90% purity). |
Note: *To ensure the purity of your primers, please call for consultation before choosing RPC purification for short-chain primers <5mer.
5. Is the 5' end of the synthesized primer phosphorylated?
The 5' end of the synthesized primer is a hydroxyl group and does not contain a phosphate group. If needed, you can use polynucleotide kinase for 5' phosphorylation, or request us to perform phosphorylation directly at the 5' or 3' end during synthesis, which incurs an additional fee.
6. What is the maximum length of a primer that can be synthesized?
The longer the primer, the higher the probability of issues occurring. Unless there is a special requirement, we recommend that the synthesized fragment length should not exceed 80mer. Based on current primer synthesis efficiency, the percentage of full-length primers in an 80mer crude product will not exceed 40%, and subsequent processing will result in a significant loss, leading to a very low final yield. GenScript can synthesize primers up to 150 bases in length.
7. Why is the fee for long-chain primers higher than for short-chain primers?
Synthesizing long-chain primers requires more reagents than short-chain primers, and the recovery yield is relatively lower, especially for primers with lengths greater than 90 bases. The increase in production cost leads to a higher price.
8. What is the quality standard for judging delivered primers?
The synthesized primers match your ordered sequence exactly. Through an optimized ordering system, an fully automated sequence import system, and a strict quality control system, we ensure that the synthesized sequences are accurate and error-free.
9. What should I do if sequencing after cloning the PCR product reveals that the primer region does not match the synthesized sequence?
In most cases, these are errors introduced during the PCR amplification and cloning processes. Should you encounter this situation, please:
- Pick and sequence alternative clones, as there is a possibility of finding a correct clone.
- You may request us to re-synthesize the primers free of charge.
10. What causes mutations found in the primer during sequencing?
Primer synthesis is a multi-step chemical reaction. The synthesis efficiency of each step is at most around 99%, making byproducts unavoidable. The longer the chain, the higher the cumulative mutation frequency. When performing cloning and sequencing after your PCR amplification, we offer the following recommendations to save time and improve the success rate:
- After detecting positive clones, please prepare cultures for 2-3 positive clones, and try to submit 2 or more clones for sequencing. This will greatly increase the success rate and save considerable time.
- Alternatively, you can submit 1 clone for sequencing first, while storing the remaining two clone cultures in the refrigerator at 4°C. If an individual point mutation or deletion occurs, immediately submit the remaining two clones for sequencing.
- This approach yields an extremely high probability of obtaining the correct sequence, avoiding a series of experimental operations like re-PCR, ligation, cloning, and screening, thereby shopping substantial time.
If you find mutations in the primer region across 2-3 or more clones, and it is confirmed to be caused by the primer itself, we will immediately arrange an expedited, free re-synthesis and deliver it to you as quickly as possible.
Primer Synthesis and Purification
Primer Synthesis, Storage, and Usage
Primer Design and Modification
