Question on the chemistry on amplicon preparation

[WSL]

Dear all,

I am working on The Universal Tailed Amplicon Sequencing but I am stuck in understanding the fundamentals of the Titanium Chemistry in Library preparation.

From Roche Handouts (Guidelines for Amplicon Experimental Design), there is a discrepancy between the versions:

May 2010
The 3’-portion of each primer is designed to anneal with the universal tails added to either side of the target of interest on the DNA sample. Their sequence, therefore, must be the complement of the Univ-A and Univ-B sequences. They are marked as Univ-A’ and Univ-B’, and shown on {indigo} or {blue} background in Figure 7.

Mar 2012
The 3’-portion of each primer is designed to anneal with the complementary strand of the universal tails added to either side of the target of interest on the DNA sample. Their sequence, therefore, must be the same as the Univ-A and Univ-B sequences (exact same sequences; not the complements nor the reverse-complements). They are marked as Univ-A and Univ-B, and shown on {indigo} or {blue} background in Figure 7.




Does anyone know which one is the correct one? If Mar 2012 version is correct, can anyone explain what is happening in the second PCR reaction and how does each strand ended up with two adaptors?


Many many thanks!!!

[ajthomas]

Your Univ-A and Univ-B primer sequences should be identical on the 5' portion of your GSP and the 3' portion of your tagging primers. The text is correct, but the figure isn't drawn exactly correct. In actuality, the forward primer will anneal with the reverse strand and the reverse primer will anneal with the forward strand.

If it's still turned around in your head, use a sequence manipulation/analysis program to create the sequence you'll have at the end, with the sequencing adapter, MID, universal adapter, amplicon sequence, etc. Then conceptually create the two (inside and outside) primer pairs you will need to amplify that sequence.

Here are a couple of the primers I use. The top one is my tagging primer, with the A sequencing adapter, MID (n's), and the universal adapter sequence. The bottom one is my GSP lined up with it.