2012-01-23.log:21:20 < nmz787> and 1 to control addition of a nucleotide... probably something that in the relaxed state, the active site was blocked/mis-aligned... and in the activated state (pulse of some chemical, light would be cool too, electrically induced ph change) it would add a nucleotide before dropping back to being inactive
2012-01-23.log:21:21 < nmz787> you could probably then move on to adding  sulfurylase and luciferase to the mix, to get a reply signal that you had a nucleotide added
2012-01-23.log:21:24 < nmz787> so maybe you eliminate the binding pocket, and the conformational change rotates an axle with a different nucleotide on 4 arms
2012-01-23.log:21:29 < nmz787> it happens naturally, a nucleotide is attracted to the template strand via the complementary hydrogen bond pattern, when it arrives the pyrophosphate (two phosphate groups linked) is cleaved from the triphosphate... this releases a lot of free energy... and the molecule takes advantage of it by changing conformations which move it down the DNA template... and the cycle repeats
2012-01-23.log:21:34 < nmz787> this video says that polymerase tests the nucleotide
2012-01-23.log:21:36 < nmz787> you might be able to replace the binding pocket with 4 nucleotides that were separate, but moved into the active site in a regulated fashion
2012-01-23.log:22:22 < nmz787> at 6-mer library, that's 4^6==4096,  4096*6 nucleotides *$0.3/bp for primers
2012-02-15.log:22:51 < nmz787> yashagaroth, kanzure: phosphatase and polynucleotide kinase are required as well, but that should be fine
2012-02-16.log:20:45 < nmz787> i forgot to add "remove polynucleotide kinase"
2012-02-16.log:21:50 < nmz787> good old natural style oligonucleotides
2012-07-10.log:18:35 < kanzure> nmz787: did you see the 5 angstrom ratcheting of DNA article? they are claiming one nucleotide resolution on moving DNA through a pore
2012-09-10.log:13:06 < nmz787> "The researchers attached a molecular motor, taken from an enzyme associated with replication of a virus, to pull the DNA strand through the nanopore reader. The motor was first used in a similar effort by researchers at the University of California, Santa Cruz, but they used a different pore that could not distinguish the different nucleotide types."
2012-11-20.log:16:26 < nmz787> 'An important design criterion for multichromophore arrays is to use structures that prevent self-quenching of electronically excited dyes by ground state dyes. This effect is seen in antibodies5,6 or oligonucleotides7that are labeled with multiple fluorophores in an effort to improve their brightness. '
2013-01-16.log:18:40 < nmz787> Nucleic acid sequencing by Raman monitoring of uptake of nucleotides during molecular replication
2013-01-21.log:16:08 < nmz787> he's checking for addition on single molecules using a fluorophore on the added nucleotide, then passing the growing strand through a nanopore past a spectrometer
2013-01-21.log:16:14 < nmz787> "in linear syntheses based on phosphoramidite chemistry, there are many potentioal sources of sequence erreor and oligonucleotide damage that are well documented. Most notable, the removeal of the 5'protecting group usually involves an acidic treatment that can removce the base, or in the case of photolabile 5'protecting group, require UV irradiation that can damage the nucleotide."... the nucleotide may fail to incorporate, nearly all the organic an
2013-02-02.log:23:49 < nmz787> a significant error contributor is damage to early-added nucleotides
2013-02-08.log:22:59 < nmz787> 'the incoming nucleotide dunks into the polymerase!'
2013-02-12.log:14:20 < nmz787> "because its recipes minimize theuse of highly error-prone (and expensive) synthetic oligonucleotides (the greatestsource of non-user error in gene assembly)"
2013-07-11.log:11:59 < nmz787> huh, so phosphoramidite nucleotides are cheaper than triphosphate nucleotides
2013-07-13.log:13:21 < nmz787> and a rich nucleotide soln flowing through
2013-07-13.log:13:25 < nmz787> and then we could use tdt and a nano-sieve to just pump in really dilute as hell (1 nucleotide per lot of channel length)
2013-07-30.log:15:34 < nmz787> kanzure: the 5' OH looks exactly like water, thus the need for everything to be super dry of H20, else 'poser nucleotide' water molecules will react
2014-12-26.log:14:13 < nmz787> really though, if you can detect with enough sensitivity, you can just control the flow to allow only a single nucleotide in
2014-12-26.log:14:14  * nmz787 thinks nanochannel and impedance spectroscopy with a dilute solution of nucleotides
2014-12-26.log:14:37 < nmz787> especially regarding nucleotides or polynucleotides
2014-12-26.log:14:45 < nmz787> so now instead of flowing in single nucleotides to tDt, you're flowing in single tRNAs to a ribosome... and hope that choking is OK and that the protein doesn't fall out
2015-01-08.log:15:35 < nmz787_i> these are quite similar concepts: " nucleotide gun made out of a nanotube pointed at the finger domain of some DNA polymerase+* single-polymerase water droplet & add in a single dNTP at a time"
2015-01-09.log:19:22 < nmz787> ... restriction endonucleases, ribonucleases, DNA polymerases, glucose oxidase, laccase, cells, viruses, proteins, peptides, small molecules, drugs, dyes, amino acids, vitamins, antixoxidants, DNA, RNA, RNAi, lipids, nucleotides, aptamers, carbohydrates, chromophores, light emitting organic compounds such as luciferin, carotenes and light emitting inorganic compounds, chemical dyes, antibiotics, antifungals, antivirals, light harvesting ...
2015-01-11.log:12:26 < kanzure> nmz787: any opinions about the idea regarding directed evolution of extracellular cell-assisted oligonucleotide synthesis?
2015-02-03.log:20:22 < nmz787> http://diyhpl.us/~nmz787/pdf/Next_generation_1536-well_oligonucleotide_synthesizer_with_on-the-fly_dispense.pdf
2015-02-26.log:00:22 < nmz787> Quantum Simulation Study of DNA nucleotide Thymine for use
2015-07-02.log:10:56 < nmz787_i> "The characteristic time of the nucleotide incorporation signal and the number of sensors per array may dictate the data throughput requirements. Data acquisition of 20 independent channels at sampling rates of greater than 1 kHz is well within the capability of commercially available, data acquisition (DAQ) cards. Thus all 20 sensors can be monitored simultaneously, with individual trans-impedance amplifiers, multipliers, and filters as needed
2015-07-02.log:10:57 < nmz787_i> "The NanoNeedle and NanoBridge sensors may be used to detect the incorporation of a nucleotide by DNA polymerase using a clonal DNA template. In an exemplary process, DNA templates were attached to magnetic beads, either via a streptavidin biotin linkage or directly conjugated, and hybridized to the sequencing primer. After pre-incubation of these beads with DNA polymerase, they were introduced into the sensor device followed by the delivery of
2015-07-02.log:10:57 < nmz787_i> number of DNA templates as well as polymerase, nucleotide and salt concentration.For proof of concept studies, both sensor type chips were fabricated with an array of functional sensing units (20 to 100 for initial design and 2 million or more in the next step) in a microfluidic channel, for example a PDMS based one. With respect to the chips without integrated magnets within the sensor array, removable magnets underneath the sensor chip were
2015-07-06.log:10:41 < nmz787_i1> just some wires to sense the nanopore for when a nucleotide passes through
2015-07-06.log:10:47 < nmz787_i1> so you can know when to turn off the nucleotide pump
2015-07-06.log:10:50 < nmz787_i1> the HO on water looks like the HO on the 2' site on the nucleotide rings, that's why it lowers efficiency... so anything with a similar HO can have that effect
2015-07-06.log:11:20 < nmz787_i1> I'd do the actual micro and nano device (on-chip peristaltic pumps, some mixing areas, a nanopore for each nucleotide, a chamber for tdt, a nano-lattice/pore-matrix to retain tdt so waste can flow, some purification areas)
2015-07-07.log:11:31 < nmz787_i> "The width of a single DNA molecule is approximately 22 to 26 Angstroms and the length of one repeating nucleotide chain link (phosphate, sugar, base) is about 3.4 Angstroms. Around 10.4 nucleotide units are required to complete one full twist of the DNA helix."
2015-07-07.log:11:31 < kanzure> one thing that i might accept from nmz787_i is if he claims something about a single-molecule-wide nanochannel that you pump the nucleotides out of.
2015-07-07.log:11:32 < nmz787_i> so 2.2 to 2.6 nm for 2 nucleotides next to each other, long ways
2015-07-07.log:12:22 < nmz787_i> but then you'd need some translator to real nucleotides
2015-07-13.log:08:49 < kanzure> nmz787_i: show me a single-nucleotide pump, or single-molecule pump from the literature, really.
2015-07-13.log:17:17 < nmz787_i> even polymerase is a sort of nucleotide pump
2015-07-13.log:17:33 < kanzure> nmz787_i: one way to do a nucleotide pump would be the following.... get a protein that attaches to a single nucleotide, make the protein super massive and large, then manipulate that protein instead of the nucleotide. this can also work for other types of tags.
2015-07-13.log:17:38 < nmz787_i> and also a way to way the pattern with nucleotides
2015-07-15.log:17:13 < nmz787_i> kanzure: can you grep the logs for my name and the word nucleotide... there is some modified nucleotide that I've mentioned before, but I can't remember the prefix/suffix