A-base addition

[seqgirl123]

In the polishing step for library preparation, there is a step using Klenow exo- polymerase and dATP, which will add one A-base to the 3' end of blunt ended DNA.

My question is how does only one A-base end up getting added onto the 3' end and not two or three?

[seqgirl123]

does anyone know the answer to this?

[shurjo]

If recessed 3' ends are present, Klenow exo- fills them in and leaves a single base overhang in ~50% of cases. Given the blunt ends from the previous step in the Illumina protocol, it simply adds a single A to the 3' end.

[seqAll]

Thanks shurjo, that was very helpful!

Does it mean that, in the Illumina protocol, Klenow exo- adds a single A to ~50% of the blunted ends?

[seqgirl123]

What are the other 50% cases about?

Sometimes it will not add at all correct? Will it ever add 2 or more A' bases?