basic question from a newby in sequencing

[joskee]

No, that's the amplification primer added while setting up the emPCR. The biotinylated primer used for enrichment is bound during the enrichment procedure, after the melt step and before adding the enrichment beads (the brown magnetic streptavidin-coated beads). The DNA beads are initially coated with just oligonucleotides.

Oh ok.

So the first primers are the oligonuceotides coated on the beads and the second primer (to make a copy of the initial template DNA from the complementary DNA bound on the bead) is the amplification primer.

And then after amplification, you melt the DNA to get ssDNA again, add the biotinylated primer (which is complementary to the DNA on the beads) and then add the streptavidin beads to enrigh.

I got it know.
It was a bit complicated to completely understand it. I didnt find the manual clear enough because they refered to later steps too early etc.
Also its not said very cleary they use an amplification primer, they immediately speak about the enrichment primers etc.. very confusing.

Thanks a lot

[ajthomas]

Ok thanks a lot.

Altough, I have one more question: the primer used to make a "second" template (from the copy made in the first round, thus amplification of the copy made the first round), is this allready the biotinylated primer for the enrichment?
ANd if those are the biotinylated primers, then the bead used during the emPCR are not streptavidine coded right? (otherwise the primers would bind the bead rather then the DNA)

No, that's the amplification primer added while setting up the emPCR. The biotinylated primer used for enrichment is bound during the enrichment procedure, after the melt step and before adding the enrichment beads (the brown magnetic streptavidin-coated beads). The DNA beads are initially coated with just oligonucleotides.

[joskee]

Yes, that's more or less correct, or at least it is for the older method of library prep. The rapid library prep protocol actually produces double standed library molecules, both of which can function as templates for emPCR. Those two strands are separated before making the emulsion, however, so you still end up with only a single strand of DNA per microreactor.

During the first round of emPCR, that molecule will bind to the bead and be copied, so after the first round you'll have one copy bound to the bead, and the original molecule will be melted off to go bind somewhere else. After the second round, there will be two complementary strands bound to the bead and two molecules melted off to go bind elsewhere on the bead, the original molecule and a copy of the copy made during the first round. After the third round there will be 4 molecules melted off, then 8, and so forth until finally at the end the bead will be covered with millions of complementary copies of the original molecule bound to the bead and millions of copies hybridized to those complementary copies and floating free in solution.

Ok thanks a lot.

Altough, I have one more question: the primer used to make a "second" template (from the copy made in the first round, thus amplification of the copy made the first round), is this allready the biotinylated primer for the enrichment?
ANd if those are the biotinylated primers, then the bead used during the emPCR are not streptavidine coded right? (otherwise the primers would bind the bead rather then the DNA)

[ajthomas]

Yes, that's more or less correct, or at least it is for the older method of library prep. The rapid library prep protocol actually produces double standed library molecules, both of which can function as templates for emPCR. Those two strands are separated before making the emulsion, however, so you still end up with only a single strand of DNA per microreactor.

During the first round of emPCR, that molecule will bind to the bead and be copied, so after the first round you'll have one copy bound to the bead, and the original molecule will be melted off to go bind somewhere else. After the second round, there will be two complementary strands bound to the bead and two molecules melted off to go bind elsewhere on the bead, the original molecule and a copy of the copy made during the first round. After the third round there will be 4 molecules melted off, then 8, and so forth until finally at the end the bead will be covered with millions of complementary copies of the original molecule bound to the bead and millions of copies hybridized to those complementary copies and floating free in solution.

I suppose you could just imagine that the drawing that's confusing you is just after the 2nd round of emPCR and there are two complementary copies on the other side of the bead that isn't shown. In reality, I think the key here it to just realize that this is a cartoon meant to illustrate the clonal amplification concept and isn't a 100% accurate representation of how it actually works. Similarly, the clusters shown for Illumina and the molecules shown for Helicos are too close to be resolved in reality, and the proteins and DNA molecules in the Pac Bio image should be in individual wells. The point is don't get too hung up on the details in the diagram; it's just trying to show the general idea in a simple way.

[joskee]

But you do realise that there will be thousands of copies of the same template eventually on each bead?

Yes, I understand that part.
I just dont understand where the second template DNA comes from...

As I understand it:

dsDNA with adaptors A and B, with B having a biotine on his 5' end.. meaning that this will bind the bead.
Then the DNA is denaturated, meaning that the 3'(on bead)-5' (not on bead, no biotine here) strand comes lose and is collected for purification and measurement of the concentration.

Then this purified DNA is added in a new mixture with beads, on those beads you have complementary pieces of DNA (complementary with the 3' region of the purified DNA, meaning that the 5' piece of those small DNA pieces is in the bead). ANd you should have 1 template DNA for every bead.
ANd then the emulsion PCR starts.


Or am I seeing this all wrong?

[nickloman]

But you do realise that there will be thousands of copies of the same template eventually on each bead?

[joskee]

I think they are demonstrating that each bead eventually ends up with a molecular colony of identical template strands. To the left of that they show an illustration of a single bead and a single fragment in a microreactor, which is the starting point for sequencing.

I can understand what you say, but to be honest: that picture isnt very clear then..because now it looks like there are 2 (or more) strands for each bead.. which is what you dont want.

[nickloman]

I think they are demonstrating that each bead eventually ends up with a molecular colony of identical template strands. To the left of that they show an illustration of a single bead and a single fragment in a microreactor, which is the starting point for sequencing.

[joskee]

Dear all,

I have the following question:
when working with 454 and more specific the emulsion PCR, it is important that you only have 1 template DNA for each bead, right?

Can someone explain to me then why in the following picture (article from nature, http://www.nature.com/nrg/journal/v1...rg2626_F1.html, first part of the picture or see the attachment) you see 2 template DNA strands in the box with emulsion PCR. THis does not seem logical.

I know its possible to have 2 strands, but then something went wrong with the preperation of the emulsion PCR, I doubt they are showing this here.

thanks in advance