Enzyme-mediated oligonucleotide synthesis

[Bryan Bishop]

Here's a method that uses terminal deoxynucleotidyl transferase (TdT) to replace some steps in phosphoramidite oligonucleotide synthesis:

http://2014.igem.org/Team:Cooper_Union/TdT_project

"""

In the general scheme shown below in Figure 1, an incoming 3'-RPdNTP (reverse protective group dNTP) is added to a free 3'OH of a growing DNA chain by the enzyme TdT at 37° Celsius. This is followed by a wash step where unincorporated 3'RPdNTPs and PPi (pyrophosphate) are removed. For heat labile 3'-RPdNTP’s deprotection is achieved by raising the temperature to 95° Celsius, while photolabile 3'-RPdNTP’s are deprotected by pulses of ultra-violet light. This is followed by another wash step where the decoupled protective groups are removed, thereby resetting the system to begin another cycle of addition.

"""

- Bryan
http://heybryan.org/
1 512 203 0507

[lesnik...@gmail.com]

This method really works or is it a theory? Is it possible to use it directly at the moment?

четверг, 25 декабря 2014 г., 23:35:56 UTC+3 пользователь Bryan Bishop написал:

[Bryan Bishop]

On Thu, Mar 10, 2016 at 2:31 AM, <lesnik...@gmail.com> wrote:

This method really works or is it a theory? Is it possible to use it directly at the moment?

They seemed to have tried it. I have CC'd Oliver Medvedik, who I think should have more information about the TdT-mediated oligonucleotide synthesis procedure. 

Oliver: For context, this was an email I received recently through the enzymaticsynthesis mailing list, https://groups.google.com/group/enzymaticsynthesis

[oli...@genspace.org]

Yes. We are working on it. The next few months we'll be performing some critical experiments. It still needs work.

[Oliver Medvedik]

Well it is still slow going, but I anticipate that keeping error rates down will be key, of course. Hopefully we can tackle that at many fronts. But first we have to establish the error rate which I thought we would have accomplished by now but haven't quite gotten there. We are shifting to UV labile protective group nucleotides, which have been very difficult/expensive to purchase until recently. We are hoping that the kinetics of decoupling will be much faster than the heat labile ones. 

On Fri, Mar 18, 2016 at 1:58 PM, Алексей Киселёв <lesnik...@gmail.com> wrote:

Bryan Bishop, thank you for help.

Oliver Medvedik, unfortunately, your labnotes ends in September 2014 (http://2014.igem.org/Team:Cooper_Union/Notebook/TdT_September) and I thought that the experiments have ended unsuccessfully. I am glad that it is not.
Can you tell us more about the problems that you have encountered? The theoretical model is understandable, but there are many pitfalls in practice. Can you tell us about this? What problems I met when I start experiments on TdT- oligonucleotide synthesis?

суббота, 12 марта 2016 г., 16:45:43 UTC+3 пользователь oli...@genspace.org написал:

Yes. We are working on it. The next few months we'll be performing some critical experiments. It still needs work.

--

Oliver Medvedik, Ph.D.

Director of Scientific Programs

Genspace NYC

33 Flatbush Ave.

Brooklyn, NY 11217

[Алексей Киселёв]

Bryan Bishop, thank you for help.

Oliver Medvedik, unfortunately, your labnotes ends in September 2014 (http://2014.igem.org/Team:Cooper_Union/Notebook/TdT_September) and I thought that the experiments have ended unsuccessfully. I am glad that it is not.
Can you tell us more about the problems that you have encountered? The theoretical model is understandable, but there are many pitfalls in practice. Can you tell us about this? What problems I met when I start experiments on TdT- oligonucleotide synthesis?

суббота, 12 марта 2016 г., 16:45:43 UTC+3 пользователь oli...@genspace.org написал:

Yes. We are working on it. The next few months we'll be performing some critical experiments. It still needs work.

[Алексей Киселёв]

Bryan Bishop, thank you for help.

Oliver Medvedik, unfortunately, your labnotes ends in September 2014 (http://2014.igem.org/Team:Cooper_Union/Notebook/TdT_September) and I thought that the experiments have ended unsuccessfully. I am glad that it is not.
Can you tell us more about the problems that you have encountered? The theoretical model is understandable, but there are many pitfalls in practice. Can you tell us about this? What problems I met when I start experiments on TdT- oligonucleotide synthesis?

суббота, 12 марта 2016 г., 16:45:43 UTC+3 пользователь oli...@genspace.org написал:

Yes. We are working on it. The next few months we'll be performing some critical experiments. It still needs work.

[Nathan McCorkle]

On Friday, March 18, 2016 at 1:01:17 PM UTC-7, Oliver Medvedik wrote:

Well it is still slow going, but I anticipate that keeping error rates down will be key, of course. Hopefully we can tackle that at many fronts. But first we have to establish the error rate which I thought we would have accomplished by now but haven't quite gotten there. We are shifting to UV labile protective group nucleotides, which have been very difficult/expensive to purchase until recently. We are hoping that the kinetics of decoupling will be much faster than the heat labile ones. 

using photo-labile nucleotides seems counter-intuitive with a tDt based system... shouldn't the enzyme be doing all the work for you? I have been working on some nanofluidic ideas to allow addition of single nucleotides at a time, I wonder how different your teams implementation is from what I'm imagining.

[Oliver]

Its to stop the reaction after one addition. 

Sent from my iPhone

[Алексей Киселёв]

Oliver Medvedik, initially you would like to use heat-labile protecting group. I like this idea because there this option are now commercially available. It looks simple and affordable for ordinary biohacker. UV method is not yet available for all. Do you have any data on the effectiveness of heat-labile protecting group method? What is the error rate?

[Oliver Medvedik]

The problem with the heat labile nucleotides that we had is that it takes way too long during each step. 

On Sat, Mar 19, 2016 at 6:11 AM, Алексей Киселёв <lesnik...@gmail.com> wrote:

Oliver Medvedik, initially you would like to use heat-labile protecting group. I like this idea because there this option are now commercially available. It looks simple and affordable for ordinary biohacker. UV method is not yet available for all. Do you have any data on the effectiveness of heat-labile protecting group method? What is the error rate?

[Алексей Киселёв]

How much time was required? 10 minutes or an hour?
Why is this happening? PCR using the same 3'-protected nucleotides occurs rapidly. Perhaps the increase in concentration of TdT will make synthesis faster?

Your experiments are very important for DiyBio community. The need to order DNA synthesis in third-party labs - the bottleneck of biohacking. This is the same as if the programmer had each time to send the source code of the program to another company, wait a few days and pay money to compile the code. Absurd.