lodging it in a pore wouldn't work, it needs to move about to interact with the fairly short seed oligos you'd start with.
On Saturday, July 16, 2016 at 5:11:27 AM UTC+1, Nathan McCorkle wrote:
-- In an effort to improve the method by finding a way not dependent on special reagents like cleanamp dNTPs I've been looking at synthesis by sequencing type methods. Consequently I'd like to propose yet another protocol.
1. a oligo sequence we will call A is introduced to TdT and ordinary dNTPs
2. the resulting extended oligos are separated by length
3. the oligos are introduced to micro beads suitable for manipulation with optical tweezers covered with covalently bound (at the 5' end) oligos of the A sequence and a second sequence we shall call B. By shear chance a small number of the oligos will have the B' sequence (we use ' to indicate the complimentary sequence) and will anneal to a small fraction the beads.
4. beads will be separated into wells and subjected to PCR.
5. both A and B sequence oligos bound to fluorophores at the the 5' end are introduced and used to mark those beads which are moved into a separate pool and washed clean on the tagging oligos.
6. a super thin layer of metal is evenly deposited on an optically flat side of a prism and A and B sequence oligos are covalently bound to the surface at the 5' end in spall regular patches.
7. the separated micro beads are deposited on the surface one on each patch and PCR takes place after which the beads are discarded.
8. the surface is washed and placed in a surface resonance imaging frame and a reference reading of the surface is taken.
9. primer A (or B) is added and allowed to anneal, a second reference image is taken.
10. a PCR mix containing only one nucleotide (dA, dT, dC or dG) is added and extension takes place (but no heating occurs)
11. the surface is washed and dried and another reference reading is taken, the difference in the surface plasmon resonance effect between rounds should indicate how many nucleotides have been added.
12. 10 and 11 are repeated with a different nucleotide (dA, dT, dC or dG) in cycles until no more extension takes place.
13. for extra proof reading 10 to 12 can be repeated with the alternate primer (B or A) to read the complimentary sequence.
We now have a metal surface with many patches with bound oligo nucleotides of the sequences metal-5'-ANB'-3' and metal-5'-BN'A'-3' and we know what the sequence N is for each patch. if we have enough patches we likely have every short sequence posable. This is a reusable resource. this metal surface can be used to synthesis DNA sequences by transferring the sequence back to micro beads (coated in A and B oligos) placed on it by using PCR. These beads can be moved and brought together in sequence, treated with type type iis restriction endonuclease specific to the A or B sequences to release the bound sequence where necessary or leave only the sense sequence intact for annealing to other sequences for ligation or PCR. By these methods longer sequences could be assembled.
Of course this depends on the surface plasmon resonance being sensitive enough to detect a single added nucleotide Since all SPR really cares about is the mass on the surface having lots of A and B primers initially bound on the metal should give a suitably strong SPR effect. ... I hope. I'm no SPR expert.
On Saturday, July 16, 2016 at 5:11:27 AM UTC+1, Nathan McCorkle wrote:
On Fri, Jul 15, 2016 at 3:47 PM, CodeWarrior <code.w...@gmail.com> wrote:
> I'm curious how you would exploit the nanopore to achieve synthesis?
oh, just a way to retain something like tDt or other enzyme for
repetitious reactions, instead of tethering it. A chunk of rock (the
silicon nitride with holes in it) seems like less variant/dynamic than
a coupling system in use (i.e. we don't have to do any covalent
chemistry)... plus it doesn't go bad sitting on the shelf like
biotin/streptavidin coupling kits might.
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