Questions: The picture and the legend below describe how TdT is used to extend oligonucleotides in two steps

The picture and the legend below describe how TdT is used to extend oligonucleotides in two steps

Solution

Solution Steps

Step 1: TdT binds to the DNA primer

The process begins with terminal deoxynucleotidyl transferase (TdT) binding to the 3' hydroxyl end of a DNA primer. The primer acts as a starting point for the extension. TdT is shown as a gray Pac-Man shape. The primer is shown as a series of black and gray rectangles representing nucleotides.

Step 2: Extension with a tethered dNTP

A modified deoxynucleotide triphosphate (dNTP) is added. This dNTP is tethered, meaning it has a special chemical modification. TdT catalyzes the addition of this tethered dNTP to the 3' end of the primer, extending it by one nucleotide (+1 nt). This is labeled "1. Extension." This results in the formation of the TdT-DNA complex. The byproduct of the extension is pyrophosphate (PPi).

Step 3: Deprotection of the added nucleotide

A cleavage reagent (such as DTT/βME, light at 365-405 nm, or a peptidase) is used to remove the tether from the newly added nucleotide. This exposes the 3'-OH group, making it available for the next round of extension. This is labeled "2. Deprotection."

Final Answer

The cycle then repeats, allowing for the controlled addition of multiple nucleotides to the DNA primer, one at a time. TdT adds a tethered nucleotide, the tether is removed, and then TdT adds another tethered nucleotide. This cyclical process enables the precise extension of the DNA primer to a desired length.

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