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
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
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