Comment by daemonk
7 hours ago
Pretty cool technique using complementary overhangs and toehold sequences to generate a 3-way heteroduplex, ligate knick, and then remove barcode duplex.
They don't give much details on how the barcode duplex is removed though. I guess ultimately the barcode duplex strands can just be melted off and the ligated strand can be used to template off of.
If this can be made into an easy to use kit, can really make vector generation much easier and hopefully not locked into proprietary systems.
I can imagine a company that bioinformatically generates libraries of common long oligos with corresponding barcode and allow end-users to select oligos to modularly ligate together in a one pot reaction. Cool stuff.
We’ve been able to do this type of nucleotide 3D engineering for a while. I used to use large DNA branched complexed fluorophores to label cDNA back when I was in grad school. They were more or less mixed of DNA that self assembled into larger hairballs.
But branched DNA is really interesting. It’s a bit hard to get my head around. We spend so much time thinking about DNA in the 2D sequence sense, it’s easy to forget that it exists in 3D space.
I’m honestly not sure how different this really is to the traditional ways of doing this (with custom oligos). The common set of large self-hybridizing oligos is definitely easier, but you still have to have compatible tag overhangs between your two fragments. Meaning, it isn’t quite as universal and you’ll still need work to pair the fragments together. But where I think it might be useful is if you have a set of common hybridizing pairs that can be easily located onto the custom flanking oligos. You’ll still need some sequence analysis to get your custom oligos, but it would make the process more “standardized”.
I think the main bonus here is the self correcting selection… that you only end up with matching pairs linking together, so you could really have a mix in a one tube reaction that links many kilobase fragments together. That’s quite nice. And useful. And still cool.
One thing that is interesting is that this is another step towards getting the “writing” step of DNA analysis better. For the past 50+ years, we’ve developed all sorts of tools for reading DNA. It’s only really been the past 20-ish or so that we’ve had tools for writing. And now we can write longer chunks. That’s all a good thing.
Not sure I think it’s revolutionary (yet), but that’s a university PR release for you! I’m still thinking about the paper.
> using complementary overhangs and toehold sequences to generate a 3-way heteroduplex, ligate knick, and then remove barcode duplex
At first I thought this was about olympic figure skating, but after a bit of googling I think:
Complementary overhang - https://en.wikipedia.org/wiki/Sticky_and_blunt_ends
Toehold sequences: https://en.wikipedia.org/wiki/Toehold_mediated_strand_displa...
Ligate (ligase?) knick (nick?) - https://en.wikipedia.org/wiki/Nick_(DNA)
Barcode - https://en.wikipedia.org/wiki/DNA_barcoding
Heteroduplex - https://en.wikipedia.org/wiki/Heteroduplex