Adapter ligation

As explained in the A-overhang 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.

The adapter is a short complementary piece of double-stranded DNA (see figure below). One strand has a T-tail at the 3' end, matching the A-overhang on the DNA, and assuring one of the two strands of the adapter (here in blue and orange) will be present at the end of each strand of the DNA sample.

Functions of adapters

The adapters have important functions in Next Generation Sequencing:

Primers: The adapters serve as a universal primer-docking site for the PCR-amplification. Since we do not know the sequence of our DNA sample (this is the purpose of sequencing!), we cannot design specific primers for PCR. The adapters provide here a known template for all different DNA strands, so that we only need to use 2 sets of primers to be able to amplify all the different small DNA molecules in the sample.

Flow cell docker: The adapters also help these DNA molecules to dock/attach to the flow cell in the sequencing machine. The flow cell is covered with many short DNA molecules that are complementary to the adapter sequence. When DNA molecules are pumped into the flow cell, the adapters will bind specifically to these fixed short DNA molecules. This is essential for Cluster generation to take place.