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corknut1 t1_iy1q8re wrote

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

The practical limit is a multiplicative function of the error rate of the chemistry involved.

If the %succes of adding a new base to the growing chain is X, and the length of the chain is N, the overall % of success for a given length is X raised to the power of N.

So for example;

If you have a 98% success rate of adding a new base to the growing chain (ie 98% of the millions of chains you are extending successfully extend),

by the time you are adding base 100, you have 98%^100 or ~13% of the original starting material as 100 base long oligonucleotides.

The shorter chains are discarded during a purification step after you've finish the addition of bases.

Practical limit of synthesis length is usually under 100 for this reason, but it is then possible (as someone mentioned in another comment) to join these together in a separate chemical process (ligation, not to be confused with the surgical definition)

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theartificialkid t1_iy1vnzi wrote

Can you use PCR to reset? (ie take your small yield of correct sequences and multiply them so you can start again at the top of the yield drop-off curve with part of your sequence already in place)

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InaMellophoneMood t1_iy1ymsr wrote

Why would you do that when you can just stitch together many chunks using modern assembly techniques? That way you can use synthesis in the cheap, high yield part of its curve, and plasmid replication/purification to yield large quantities of your sequence for even cheaper. Fussing with super long synthesis with the flaws of existing chemistries doesn't make sense when it's more time and labor intensive than assembling them from medium length synthesis.

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corknut1 t1_iy4z7or wrote

Not really - the initial base needs to be attached to a substrate (e.g. CPG) or support. It's only removed from this substrate at the final step once you have finished extending the chain.

Think of the support as the thing that keeps your DNA in the bottle when you're doing the chemistry; during the synthesis you're repeatedly adding chemicals then washing them away.

If your DNA product isn't firmly attached to something during this process, it's going to get washed away too.

It's conceivable you could remove the DNA from the support, capture it, amplify it with PCR, then reattach to support to continue the extension, but the re-attach part would be very difficult - you'd be dealing with a long floppy chain and trying to attach one end to a solid anchor via some unknown complex chemistry. There would be side-products, loops, breaks, etc. to deal with. Someone has probably tried it, but it's not something I've ever encountered.

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