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Oligos are short-chain molecules, typically containing 20-30 nucleotides that can bind to specific DNA or RNA sequences through complementary base pairing, thereby guiding the polymerase to amplify DNA at that sequence. The core principle ofoligo synthesis is the reaction of nucleotides with specific chemical reagents on a solid-phase carrier, progressively linking them to form the desired nucleotide sequence.
The main methods for synthesizing DNA oligonucleotides are phosphoramidite synthesis, solid-phase synthesis, enzymatic synthesis, and rolling circle amplification.
Here’s a breakdown of each method and their differences:
|
Phosphoramidite Synthesis |
Solid-Phase Synthesis |
Enzymatic Synthesis |
Rolling Circle Amplification |
|
|---|---|---|---|---|
|
Principle |
Using a stepwise assembly of nucleotides on a solid support. |
It refers to the attachment of the growing DNA strand to a solid support while nucleotides are added sequentially in a protected form. |
Utilizing enzymes to synthesize oligonucleotides. |
Amplifying circular DNA templates by repeatedly synthesizing new strands from a primer that binds to the circular DNA. |
|
Pros |
High efficiency, well-established for synthesizing oligonucleotides up to about 200 nucleotides, and flexible for designing custom sequences. |
Allows precise control over the synthesis process and is the backbone of many automated oligonucleotide synthesis platforms. |
Less expensive than chemical synthesis and can produce longer sequences. |
Capable of synthesizing longer sequences and can be highly efficient. |
|
Cons |
Expensive and requires specialized equipment. |
As with phosphoramidite synthesis, it is costly and can be complex for longer sequences. |
Low flexibility, Limited by the fidelity and efficiency of the enzymes. |
Typically more complex and requires specific enzymes and conditions. |
|
Applications |
Widely used in research, diagnostics, and therapeutic applications for generating short DNA fragments (e.g., primers, probes). |
Used for custom oligonucleotide synthesis, especially when high specificity and purity are required. |
Used for constructing longer DNA fragments, cloning, and in certain synthetic biology applications. |
Primarily used in molecular diagnostics and research for amplification of DNA sequences and generating long DNA strands. |
Among them, thephosphoramidite method is the most commonly used method for synthesizing DNA oligonucleotides. It utilizes phosphoramidite monomers, which, under the action of activators such as tetrazole, undergo coupling reactions with nucleotides on a solid-phase carrier to form phosphotriester bonds. This method has the advantages of mild reaction conditions, high synthesis efficiency, and high product purity, making it widely used in the synthesis of DNA oligonucleotides.
DNA oligonucleotides Synthesis | Phosphoramidite
Step 1 (Detritylation)
The cycle is initiated by removal of the 5'-DMT (4,4'-dimethoxytrityl) protecting group of the solid-support-linked nucleoside (contains the terminal 3' base of the oligonucleotide).
Step 2 (Coupling)
Once the DMT has been removed, the free 5'-OH of the solid-support-linked nucleoside is able to react with the next nucleoside, which is added as a phosphoramidite monomer.
Step 3 (Oxidation)
The phosphite triester formed during the coupling reaction is unnatural and unstable; therefore, it must be converted to a more stable phosphorus species prior to the start of the next cycle. Oxidation converts the phosphite triester to the stable phosphate triester.
Step 4 (Capping)
Since 100% coupling efficiency is impossible, there are always some solid-support-linked nucleosides with unreacted 5'-OH. If not blocked, these hydroxyl groups will react during the next cycle, and hence, lead to a missing base. Capping is required to prevent shorter accumulation.
Importance of primers in molecular biology experiments
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PCR Amplification: Primers are key components in the PCR amplification process, they bind to specific regions of the template DNA and direct DNA polymerase to synthesize the complementary strand along the template strand. Without proper primers, PCR amplification will not be possible.
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Gene cloning: In gene cloning, primers are used to amplify and insert target DNA fragments into a vector, allowing for gene replication and expression.
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Gene expression analysis: By designing primers for a specific gene, the expression level of the gene can be amplified and detected to understand the function of the gene in the cell or tissue.
Precautions for oligo synthesis
Primer design: Primer design is one of the key steps in the primer synthesis experiment. Primer length, GC content, specificity and other factors should be considered to ensure that the primer has a good amplification effect.
Synthesis method: oligo synthesis usually adopts automated synthesis method, adding bases step by step on the chemical synthesizer to construct the required sequence. During the synthesis process, attention should be paid to the reaction conditions, reagent quality and other factors to ensure the quality of primers and synthesis efficiency.
Oligo purification and detection: The oligos after synthesis need to be purified and detected to improve the purity and amplification effect of the oligos. Commonly used purification methods include chromatography, affinity chromatography, etc.; while detection methods include gel electrophoresis, fluorescence quantitative PCR, etc.
Synbio Technologies |Oligos Synthesis
At Synbio Technologies, our advanced oligo synthesis platform is equipped with state-of-the-art synthesizers and supported by a team of experts in synthesis and modification technologies. This allows us to deliver high-quality oligonucleotide products to researchers and industrial customers worldwide. We offer a broad range of oligonucleotide synthesis options and provide high-throughput oligo library and oligo pool synthesis for large-scale projects.
