emulsion PCR in detail explained ?

[avierstr]

Hello,

I'm making a presentation about Next-Gen sequencing and I'm trying to make it as understandable as possible. I'm making a drawing to explain emulsion PCR, but I can't find the detailed steps what is happening in the reaction (something like what I did voor PCR : principles of pcr)(Google fails me here).

Most of the explanations on the internet are like this : Emulsion PCR, so you start with a bead with one ssDNA, and ends with a bead full of ssDNA. I'm missing the steps between those two...

So, this is what I think is happening : (hopefully someone can correct or confirm this :

(assume : adaptor A on one side of the ssDNA (anneals to bead), primer P on the other site of the ssDNA)

1. One ssDNA strand (forward strand) anneals to the complementary adaptersite A attached to the bead.

2. Extension starts from the bead (on the complementary adapter) and copies the template. Now we have a forward and a reverse strand.

3. Denaturation : the original ssDNA strand (forward strand) releases from the bead. The copy (reverse strand) is attached to the bead.

4. Annealing : the original ssDNA strand (forward strand) anneals to an other adapter on the bead. Also, a primer anneals to the reverse strand which is stuck to the bead (primer anneals on the side away from the bead)

5. Extension : forward strand starts from the bead and copies towards the primersite, reverse strand start from the primer and copies towards the bead

6. Back to 3 and this goes on for XX cycles.

Is my assumption correct that there is only one "free" primer in the mixture (the other primer is the one attached to the bead)

Thanks,
Andy

[krobison]

Seems about right. The two bits missing from your description are (1) The beads are enclosed in an emulsion, so no cross-talk between beads and (2) The emulsion is prepared at a dilution so that a sizable fraction of emulsified beads have only a single template in the same compartment. This is, of course, stochastic -- some will have multiple and some none, and that must be dealt with later in some way.

One of the original executions of emulsion PCR was called "molecular BEAMing", and you may find that a useful serch term (e.g. http://www.pnas.org/content/100/15/8817.full.pdf)

[avierstr]

Thanks for the confirmation. I was aware of your remarks. These are every presentation, but not the steps I was looking for.

Best regards,
Andy

[avierstr]

My drawing is finished. Maybe other people can use it, so, here is the link. If someone finds errors, let me know.

http://users.ugent.be/~avierstr/nextgen/emulsionpcr.jpg

Greets,
Andy

[billyhuffsmith]

The animation on the 454 site at http://454.com/products/technology.asp shows a subtly different starting point than your drawing.

In the animation, single stranded DNA is attached to beads before the beads enter emulsion droplets, so that the exact situations shown in your circles pointed to by the arrows 'start PCR' and '1. Denaturation of library fragment' never occur.

[billyhuffsmith]

Upon further consideration, the 454 animation is not internally consistent in terms of 5'-3' directionality and must not be intended to be taken literally.

[avierstr]

It took some time, but my presentation is finished. 128 slides which explains the most common platforms and how they work in detail. Applications and the complexity of data processing is presented with limited explanation. You can find it on my website :

http://users.ugent.be/~avierstr/nextgen/nextgen.html

[SEQtheTruth]

It took some time, but my presentation is finished. 128 slides which explains the most common platforms and how they work in detail. Applications and the complexity of data processing is presented with limited explanation. You can find it on my website :
http://users.ugent.be/~avierstr/nextgen/nextgen.html

Bravo!
Thanks for the informative & historical guide. I would imagine that plenty of newcomers to bioinformatics (other than myself) only have foggy ideas of why there are adapters on their reads, or why they're all the same length. Great source of context.

I know it's a year old now, but you should try to get this to show up on Google before it really becomes outdated...

[aedanr]

If you're still reading, avierstr, just want to say thanks for your really clear depiction. I just used your emulsion PCR picture in a talk - gets the ideas across really nice and clearly.

[relaswar]

emPCR animation

Great explanation. Wish Roche prepared similar animated description for the entire NGS process and presented on their website. I'll definitely use it in my future talks.....

[md.wasim]

what are limitations & disadvantages of emulsion pcr?

[md.wasim]

emPCR

what are limitations & disadvantages of emulsion pcr?

Can someone insight me on limitation part of emPCR ? Thanks in advance

[AXSILVA]

For one serving the PPase emPCR?. Forgot to put it and if not follow the preparation of the run.