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At present, the primer synthesis is basically based on the solid phase phosphite amide triester method. The synthesis of DNA fragments by phosphite amide triester method has the characteristics of high efficiency, fast coupling and relatively stable initial reactants.

Phosphamide triester method is to fix DNA on solid carrier to complete the synthesis of DNA chain. The synthesis direction is from 3 'end to 5' end of the primer to be synthesized, and adjacent nucleotides are connected by 3 '→ 5' phosphate diester bond.

Service features and advantages：

Qiangyao Bio has a variety of synthesis instruments, among which BLP platform is the leading one for high-throughput synthesis and SpectraMax is used to monitor the efficiency of synthesis in the whole process; In addition, our company also has ABI 3900 and BioAutomation, which can be synthesized on a large scale and specially used for some special purposes. In addition, the company has a variety of models of high-performance liquid chromatographs. The one-time preparation can meet the synthesis level of 50nmol-50umol, and the purity can reach more than 95%.

Strict quality control standards

With strong technical support,

The synthetic purity can reach above 95%.
Fast delivery

It can be shipped within 48 hours at the earliest,

Arrival within 1-2 days in China.
Multiple purification methods

With strong technical support,

The synthetic purity can reach above 95%.
Synthesis of various modified primers

It can provide a variety of high-quality fluorescent modifications.

Introduction and selection of purification methods

Purification Method	Purification Principle	Advantage	Shortcoming	Scope Of Application
DSL purification	Using the latest biolytic meteorological ammonolysis instrument, the primer connected to CPG is cut and eluted under the gas phase ammonolysis environment of high-purity ammonia steam mixture, which can effectively remove salt, and some impurities can be removed through gradient elution of organic solvent.	Compared with the traditional liquid phase ammonolysis, it is efficient, fast, and free of cross contamination. It is the simplest of all purification methods.	Small fragments shorter than the target fragment cannot be effectively removed, provided that the synthesis effect is good.	PCR amplification, subcloning, point mutation, whole gene synthesis, DNA sequencing, etc.
OPC purification	Use the resin with special affinity for DMT group in OPC column to retain DMT on the last base at 5 'end when synthesizing DNA fragments. After all synthetic products are adsorbed on OPC column, wash the column with dilute organic solvent. The segment with DMT has strong adsorption capacity and is not easy to be eluted. The segment without DMT has weak adsorption capacity and is eluted. Then use acid to remove DMT adsorbed on DNA on OPC column, Then elute DNA with a denser organic solvent.	Convenient and fast	Its ability to adsorb DMT specifically is limited, so it is still possible to bring in short fragments, and its load is small, especially for fragments longer than 40 bases.	PCR, the key of primer is 5 'sequence, such as restriction enzyme digestion site; Mutation at designated sites; The first strand of cDNA is synthesized when the library is synthesized; Generation of clone connector.
PAGE purification	The target sequence and failed sequence can be separated by using different mobility of DNA sequences with different lengths in gel, so as to provide high-purity target primers. It is particularly effective for the purification of long chain primers (greater than 40 mer). The company uses C18 reversed-phase column with capillary electrophoresis, and the purity is more than 85%.	The purification effect is good, especially the purification of long chain.	The degree of automation is low, the primer loss is large in the purification process, and the delivery cycle is relatively long.	The most effective method for purification of common primers above 40 mer; Used for site directed mutagenesis and cloning; Protein binding gel electrophoresis for analysis, treatment and diagnosis.
HPLC purification	HPLC is divided into reverse phase column and ion column according to the adsorption medium. The reverse phase column is separated according to the difference of hydrophobicity, that is, the longer segment with stronger hydrophobicity has stronger adsorption capacity than the shorter segment, and then peaks; The oligonucleotides on the ion column have different net charges according to different lengths. The longer fragments with high charges flow slower in the ion column than the shorter fragments with low charges, thus eluting different fragments in turn.	High degree of automation, manpower saving, good purification effect, purity can reach more than 99%, especially in the purification of labeled primers and special modified probes.	The purification amount is small, long fragments cannot be purified, and the equipment is expensive.	The purification of common primers<50 mer for site directed mutagenesis, cloning, protein binding gel migration electrophoresis analysis and therapeutic purposes; Purification of modified primers; Commercial diagnostic primers or probes.

Application of common modifiers

Phosphorylation Phosphoryl	5 'phosphorylation can be used in splices, cloning and gene construction, as well as ligase catalyzed linkage reactions. Phosphorylation is also used to prevent DNA strand elongation reaction catalyzed by DNA polymerase in relevant experiments where it can resist digestion by exonuclease.
Biotin Biotin	Biotin modification is resistant to heat and pH. Biotinylated DNA can be successfully transformed into bacteria. Primer biotin labeling can be used for non radioactive immunoassay to detect protein, intracellular chemical staining, cell separation, nucleic acid separation, hybridization to detect specific DNA/RNA sequence, ionic channel conformation changes, etc.
Digoxigenin Digoxigenin	Dixin is a kind of steroid substance separated from the plant of Digitalis pubescens. Because the flowers and leaves of Digitalis pubescens are the only natural source of this substance, the anti Dixin antibody will not bind to other biological substances. Digao is connected to the C5 position of uracil via an arm of 11 atoms. The hybridized digoxin probe can be detected by anti digoxin antibody, which is usually coupled with alkaline phosphatase, peroxidase, fluorescein or colloidal gold. Or there is no even anti digoxin antibody but even anti antibody. The probes labeled by Digao can be used for various hybridization reactions, such as DNA DNA hybridization, DNA RNA hybridization, dot blotting, clone hybridization, in situ hybridization and enzyme-linked immunosorbent assay (ELISA).
Internal amino modification	C6-dT aminolinker is mainly used to add thymine residues for internal modification. The modified amino group is 10 atoms away from the main chain, which can be used for further labeling and enzyme linking (such as alkaline phosphatase).
5 'amino modification	It can be used to prepare functional oligonucleotides, and is widely used in DNA microarray and multiple marker diagnostic systems. At present, there are two kinds of 5 'C6 amino modification and 5' C12 amino modification. The former can be used to connect some compounds that will not affect their functions even if they are close to oligonucleotides, and the latter is used to connect affinity purification groups and some fluorescent labeling, especially when the fluorescence may be quenched because the labeling is too close to the DNA chain.
3 'amino modification	It can be used to prepare functional oligonucleotides, and is widely used in DNA microarray and multiple marker diagnostic systems. At present, there are two kinds of 5 'C6 amino modification and 5' C12 amino modification. The former can be used to connect some compounds that will not affect their functions even if they are close to oligonucleotides, and the latter is used to connect affinity purification groups and some fluorescent labeling, especially when the fluorescence may be quenched because the labeling is too close to the DNA chain.
Spacer	5 '- mercapto is similar to amino modification in many aspects. Thioglycol can be used to attach various modifications such as fluorescent markers and biotins. For example, thiol linked fluorescent probes can be made in the presence of iodoacetic acid and maleimide derivatives. The mercapto modification of 5 'mainly uses 5' mercapto modified monomers (5 '- Thiol Modifier C6 CE Phosphoramidite or Thiol Modifier C6 S-S CE Phosphoramidite). After modification with 5 '- Thiol Modifier C6-CE monomer, silver nitrate oxidation must be carried out to remove the protective group (trityl), while after modification with Thiol Modifier C6 S-S CE monomer, the disulfide bond must be reduced to thiol with DTT.
Thio Phosphorothioate	Thio modified oligonucleotides are mainly used to prevent degradation by nuclease in antisense experiments. You can select full thio, but the Tm value of oligonucleotide will decrease with the increase of thio base. In order to reduce this effect, you can thio modify 2-5 bases at both ends of the primer. Generally, you can select 3 bases each of 5 'and 3' for thio modification.
Deoxyuracil DeoxyUridine，dU	Deoxyuracil can be inserted into oligonucleotides to increase the melting point temperature of double chains and thus increase the stability of double chains. The substitution of each deoxytymine by deoxyuridine can increase the double chain melting point temperature by 1.7 ℃.
Deoxyhypoxanthine deoxyInosine，dI	Deoxyhypoxanthine is a naturally occurring base. Although it is not a universal base in the true sense, it is more stable than other base mismatches when combined with other bases. The binding ability of deoxhypoxanthine to other bases is dI: dC>dI: dA>dI: dG>dI: dT Under the catalysis of DNA polymerase, deoxynhypoxanthine first combines with dC.

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