Why add 'A' to 3' end of DNA when preparing sample

Question

[897598644]

Excuse me:

The SureSelect protocol said that we should add 'A' bases to the 3' end of the DNA fragments. Could someone tell me the purpose of this procedure?

Many thanks in advance!

Answer 1

[Cliff Beall]

As far as I know, this is not specific to SureSelect, it's all Illumina ligation-based protocols.

When you ligate the sequencing adapters to the target insert, it helps get adapter to insert ligation, because the adapters have a T overhang on their 3' ends and can pair. The inserts or adapters ligate to themselves much less efficiently because they will have A-A or T-T mismatches.

[Devon Ryan]

Yeah, this is a general molecular biology technique. Whenever you need to subclone something you try to either use non-blunt restriction enzymes or use Topo-cloning (or similar), where an A-tail is used for the same reasons. Blunt ligations facilitate more non-desired ligation products.

Answer 2

[Devon Ryan]

Adding an A overhang is a standard mechanism to facilitate more specific ligations (not that the adapters also have an overhang, of a T).