Q&A Example (A Question Prof. Kalderon Addressed by Email):

Question:

I am a little confused about why blocking groups are necessary for chemical DNA synthesis.  I thought that without blocking unwanted side reactions would occur and you would get some oligos with incorrect sequences. On the other hand, without capping you would get internal base deletions.  I thought that capping and blocking were sort of the same thing.Could you explain how problems with blocking would be problematic?

Three different concepts- protection, blocking & capping (protection & blocking might be used interchangeably by some but they include two different concepts)

Protection is simple- reversible chemical modification that protects reactive groups on bases (amino groups) from being altered during oligo synthesis

Blocking; each new nucleotide to be added has a blocked 5' hydroxyl (DMT- dimethyltrityl group attached). The new nucleotide is added AFTER the growing oligo has the DMT removed to expose a 5'-OH. Hence, the new nucleotide at its 3' position adds on  via (almost) phosphodiester linkage to only the growing oligo and not to itself (5'-OH is blocked).

Capping contends with the 1% or fewer of growing oligos that fail to add to the new oligo. If nothing were done these oligos could be added to in the next & subsequent rounds of synthesis, producing an almost correct oligo, but just missing one base. Almost all impurities would therefore be just 1base deletions at various positions. These may compete well with the correct product in many applications (in some it may matter- sequencing, producing an exact PCR product; in others not- just producing a PCR product where terminal sequences are not important). Capping blocks the 5'-OH of an oligo that failed to add a new nucleotide PERMANENTLY so it is not reversed in the next step of synthesis and all oligos that fail to be extended at any step are not added to further. Hence, contaminants range in size from 1nt to just 1nt shorter than the correct oligo. Most such oligos will not hybridize to any template in subsequent applications where high stringency conditions are used. Also, the size range of contaminants makes the capped, defective oligos easier to remove by purifications based on oligo length (usually done by HPLC).