use 3' Phosphorylation

Hi there,

A 3'H instead of the 3'OH is 100% efficient to block extension! This is the principle of Sanger sequencing...

Hi... The modifications most frequently used for this purpose are the 3'ddC, 3' Inverted dT, 3' C3 spacer, 3' amino, and 3' phosphorylation.

The 3' end modifications that prevent DNA from being extended by DNA polymerase include dideoxy (ddNTP), 2',3'-dideoxy, and 3'-O-azido. Among these, dideoxy (ddNTP) has the best blocking effect, as it lacks the 3' hydroxyl group necessary for further DNA strand elongation by DNA polymerase. This absence prevents the addition of subsequent nucleotides, effectively terminating DNA synthesis. Consequently, ddNTP is widely used in Sanger sequencing and other applications where controlled termination of DNA synthesis is required, and it typically results in minimal leakage.

3' end modifications that prevent DNA from being extended by DNA polymerase are critical in various applications, such as DNA sequencing, gene synthesis, and molecular cloning. These modifications block the addition of nucleotides, effectively halting DNA polymerase activity. Here are some common 3' end modifications and their effectiveness:

1. **Dideoxynucleotides (ddNTPs)**:

- **Description**: Dideoxynucleotides lack a 3' hydroxyl group (OH) on the ribose sugar, which is essential for forming a phosphodiester bond with the next nucleotide.

- **Effectiveness**: Highly effective. ddNTPs are used in Sanger sequencing precisely because they terminate DNA synthesis efficiently.

- **Leakage**: Minimal. The lack of the 3' OH group ensures that no further nucleotides can be added.

2. **3' Phosphate Group (3'-PO4)**:

- **Description**: The addition of a phosphate group to the 3' end prevents the formation of a new phosphodiester bond.

- **Effectiveness**: Effective but can be less stable compared to ddNTPs. In some conditions, phosphatases can remove the 3' phosphate, allowing extension.

- **Leakage**: Generally low, but conditions must be carefully controlled to prevent enzymatic removal.

3. **3' Inverted Nucleotide (3' Inverted dT)**:

- **Description**: An inverted nucleotide (e.g., 3' inverted dT) has the nucleotide flipped in orientation, preventing extension.

- **Effectiveness**: Very effective. The altered structure of the inverted nucleotide makes it a strong block for polymerases.

- **Leakage**: Minimal. The unusual structure of the inverted nucleotide is not recognized by DNA polymerases for extension.

**Best Blocking Effect with Minimal Leakage**:

Among these, **dideoxynucleotides (ddNTPs)** are typically considered to have the best blocking effect with minimal leakage. The complete absence of the 3' hydroxyl group ensures that DNA polymerase cannot extend the DNA strand, making ddNTPs highly effective for applications requiring precise termination of DNA synthesis, such as in DNA sequencing.