Viewing a single comment thread. View all comments

toolemeister t1_iy1bkhy wrote

Reply to comment by YouDrink in How exactly does CRISPR-CAS9 insert new genes? by AutomaticAd1918

How is this physically done, as in equipment/machinery etc.? I understand the abstract concepts but I'm more interested in the engineering!

6

corknut1 t1_iy1rzwq wrote

It's essentially done with robotic pipettes, though the ones used in DNA synthesis are very specialized.

It can be done with hand pipettes, but it would be a slow and error prone process.

The materials are not exotic and can be purchased from chemical supply.

Typically the starting material is a (population of) a single DNA (or RNA) base bonded to a glass bead (cpg), and the chemistry is done by washing the beads with a series of chemicals, following a repeated recipe. The recipe is identical for each base added to the chain (excepting the base itself of course).

11

RoundScientist t1_iy7qqoi wrote

This is what was described to me in a lecture.
You have beads of resin which you can chemically link chemical derivatives of nucleotides to.

  1. You link your first nucleotide to the bead.
  2. You flush out excess nucleotides.
  3. You add a reactant that makes all unoccupied parts of the resin non-reactive for the kind of chemistry you're doing.
  4. You remove the protective group that prevented the nucleotide derivatives from linking up with one another.
  5. You have unprotected nucleotide derivatives chemically linked to your resin beads. You now start back at step 1 - only this time, the new nucleotides you add bind to those from the previous cycle. After all, these are the only positions left where you can have linking reactions.

And then you just loop the process and by picking the order of reagents, you determine the nucleotide sequence. The inactivation step prevents the further growth of oligonucleotides which "missed" a step.
Once you're done, you perform a reaction which splits the oligonucleotides off the beads and purify them by length. I think usually via chromatography.

Which means that you have a machine with several input liquids and valves (solutions of your reagents, solvent, one reservoir & valve for solutions of each derivative nucleotide), a reaction chamber with beads and a valve for removing liquid/trash collection.
Then it's just a matter of sucking in/out the right liquids in the right order, with long enough pauses for the reactions to take place. Possibly with heating.

1