Primer Synthesis

The key to successful sequencing is that the sequencing primer has only one binding site on the DNA template molecule. If the sequencing primer has more than one binding site on the DNA template, it will cause the primer to initiate extension at multiple sites during the sequencing reaction. This will manifest as double peaks or messy peaks on the sequencing chromatogram, making the sequence unreadable.

If the water used to dissolve the primer has a low pH or is contaminated with bacteria or nucleases, the primer can degrade. If the primer is not fully thawed and mixed before use, liquid inhomogeneity might lead to inaccurate primer addition amounts. It is recommended to aliquot primers to avoid repeated freeze-thaw cycles. Using 10 mM Tris pH 7.5 buffer to dissolve primers is recommended, as some distilled water has a relatively low pH (pH 4-5), and primers are unstable under these conditions. Another possibility is that the primer is fine, but the quality of the PCR materials, especially the template, is not identical to that used previously.

The dried product is very stable and can be stored at -20°C for 2 years without issue.Dissolved DNA should also be stored at -20°C, preferably aliquoted to avoid repeated freeze-thaw cycles. Oligo DNA in solution should be fine at room temperature for 3–4 days, but it is best not to leave it for more than a week. Its longevity is related to container and solvent sterility.

Denaturing PAGE electrophoresis must be used for Oligo DNA. Oligo DNA is single-stranded and prone to forming complex secondary structures. Therefore, during Agarose gel electrophoresis, multiple bands often appear (and Agarose gel electrophoresis cannot be used for quantification).

No, Both the 5' and 3' ends are -OH groups. If phosphate groups are required, please specify this when ordering. In this case, a phosphorylation (PO4 modification) fee will be charged.

General requirements for sequencing primers:
(1) Specific binding to the sequencing template, with no more than 4 base mismatches.
(2) Must not contain mixed bases.
(3) Length: 17–25 bases.
(4) High purity, preferably PAGE purified.
(5) Dissolved in ddH₂O, not TE buffer.

The synthesized primer matches the sequence in your order, not whether it can amplify your desired product.

If the primers in the ligation reaction lack a 5' phosphate group, the ligation efficiency is relatively low, but it may not completely fail.
If the phosphorylated product still cannot be ligated into the vector, it is necessary to check the vector digestion efficiency and review the primer design. If no issues are found, consider optimizing the primer annealing conditions and the ligation reaction system.

DNA synthesis length: 6–130 bases, RNA synthesis length: 8–35 bases.
When synthesizing long DNA sequences, it is difficult to ensure that every molecule in the product is completely correct due to limitations in synthesis and purification methods. Additionally, it should be noted that if the sequence to be synthesized is particularly challenging (e.g., containing consecutive "G"s, etc.), the synthesis yield may be relatively low, and we may charge an additional fee based on the specific situation. Sometimes, we may be unable to synthesize the ordered sequence, in which case the company reserves the right to decline the order.

Nucleic acids have strong absorption near 260 nm, while proteins absorb strongly near 280 nm. When extracting nucleic acids from biological sources, the OD260/OD280 ratio is commonly used to assess nucleic acid purity (a ratio between 1.8–2.0 is typical). This judgment is based on the assumption that the proportions of A, G, C, T in the sequence are roughly equal. However, this is not the case for synthetic DNA/RNA. The sequences are short (typically 20–30 bases), and the proportions of A, G, C, T bases can vary greatly. Due to the different molar extinction coefficients of the various bases, the OD260/OD280 ratio of synthetic DNA/RNA products differs depending on the base composition. For example, when the sequence has a high content of C and T bases, this ratio can be significantly lower than 1.8. Additionally, the sequence order of the bases also affects this ratio. Therefore, the OD260/OD280 ratio cannot be used to evaluate the purity of synthetic DNA/RNA products.

1. Generation of Mature siRNA (Double-Stranded RNA Processing)​

siRNA originates from the cleavage of exogenous or endogenous double-stranded RNA (dsRNA) by the Dicer enzyme (a member of the RNase III family):​

    Source of dsRNA: Exogenous sources include viral dsRNA (e.g., from RNA viruses) or artificially synthesized dsRNA (e.g., siRNA used in experiments); endogenous sources are rare (mostly from transposons or repeat sequences).​

    Cleavage process: Dicer recognizes dsRNA and cleaves it into short dsRNA fragments (21–23 nucleotides in length) with two key structural features: a 5′-phosphate group and a 2-nucleotide overhang at the 3′-end—these structures are essential for subsequent RISC assembly.​

2. Assembly of the RNA-Induced Silencing Complex (RISC)​

The mature siRNA duplex binds to the Argonaute (Ago) protein (the core catalytic component of RISC) and other accessory proteins (e.g., RNA helicase) to form the active RISC, with two critical sub-steps:​

    Strand separation: Under the action of RNA helicase, the siRNA duplex is unwound into two single strands:​

    Guide strand: The strand with higher stability at the 5′-end (determined by base pairing energy); it remains bound to Ago and serves as the "guide" for target recognition.​

    Passenger strand: The complementary strand of the guide strand; it is structurally unstable and is degraded by Ago’s nuclease activity, ensuring RISC only retains the functional guide strand.​

    Activation of RISC: After strand separation, the Ago-guide strand complex (active RISC) is formed, and its "seed region" (nucleotides 2–8 of the guide strand) is exposed to prepare for target mRNA recognition.​

3. Target mRNA Recognition and Post-Transcriptional Gene Silencing (PTGS)​

The core function of active RISC is to specifically silence target gene expression by recognizing and degrading mRNA, relying on base complementarity between the guide strand and target mRNA:​

    Specific recognition: The guide strand of RISC binds to the 3′-untranslated region (3′-UTR) or coding region of target mRNA through complete base complementarity (a key feature distinguishing siRNA from miRNA, which usually binds via incomplete complementarity).​

    mRNA cleavage and degradation: The Ago protein in RISC has a conserved PIWI domain (a nuclease domain) that cleaves the phosphodiester bond of the target mRNA at the site 10–11 nucleotides downstream of the guide strand’s 5′-end. The cleaved mRNA fragments are further degraded by cellular exonucleases (e.g., XRN1), preventing them from being translated into proteins.​

    Result of silencing: Since the target mRNA is degraded before translation, the expression of the corresponding target gene is significantly reduced (transcription is unaffected; silencing occurs at the post-transcriptional level).​

Key Summary​

The core mechanism of siRNA-mediated RNAi can be simplified as: dsRNA cleavage by Dicer → active RISC assembly (guide strand retention) → target mRNA cleavage via RISC → post-transcriptional gene silencing. This process is highly specific (dependent on guide strand-mRNA complementarity) and efficient (one RISC can cleave multiple mRNA molecules), making it a core tool for gene function research.