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Adapter ligation

As explained in the end-repair section, the purpose of adding adenine at the end of the DNA strand is to allow specific pairing with the thymine tail of the adapter.

At the top are two horizontal gray lines, illustrating blunt DNA. The left end of the first line is marked as the 5' end and the right end is marked as the 3' end. On the second line are the ends reversed, with the 3' end to the left and the 5' end to the right. Beneath them are another set of identical gray lines, but with a blue arrow illustrating how adenine is added at the 3' end of the blunt DNA. Adenine is marked on both gray lines as a green A. Another set of horizontal lines, illustrating the thymine tail of the adapter are colored blue and yellow and marked with a T. A blue arrow illustrates how the adenine tail of the DNA sample will bind specifically with the thymine tail of the adapter.

The adapter is a short complementary piece of double stranded DNA. One strand has a T-tail at the 3' end, and the other strand is complimentary to the first strand.

Because adenine pairs up specifically with thymine, we can be sure that one end of the DNA sample will have the blue adapter, whereas the other end will have the orange adapter.

Functions of adapters

The adapters have important functions in Next Generation Sequencing.

Primers

The adapters also serve as universal primer docking sites. It is not possible to individually design primers that match a specific DNA molecule to be able to amplify our whole genome! The adapters provide a cap for all different DNA strands so that we only need to use two sets of primers to amplify all the different small DNA molecules.

Flow cell docker

The adapters also help these DNA molecules to dock/attach to the flow cell. The flow cell is covered in many short DNA molecules that are complimentary to the adapter.

When DNA molecules are pumped into the flow cell, the adapters will recognize the short DNA molecules attached to the flow cell and bind together. This is important for cluster generation to take place.

Referred from:

Article
A-tailing