I am making some infographics of different library prep types and noticed something weird about NEB's Ultra II adapters.
Molecule 1
is the desired product of the adapter ligation step. *
indicates a phosphorothioate bond and -
indicates a normal bond. |
indicates complementary sequence through hydrogen bonding.
Molecule 2
is the product after treatment with the USER enzyme.
Molecule 3
is the result of melting molecule 2
and hybridizing the NEBNext Universal PCR Primer for Illumina.
My question is: Why does Molecule 3
have a large non-complementary region on the 3' end of the original molecule? Is that just to force Molecule 1
to have an open loop conformation so that the USER enzyme can access it better? Or maybe it is something to do with PCR kinetics?
I understand that PCR synthesizes in the 5'->3'
direction, so Molecule 3
only forms with the original adapter-ligated molecules before PCR.