PS bonds and polymerase

[biochembug]

Hi folks

I am planning to use illumina primers RP1 and RPI1 with phosphorothioate bonds between the last base and the penultimate base at the 3' end. So my eventual PCR library will contain two phoshorothioate bonds, one on each strand.

Can polymerase read through such products during cluster formation or regular PCR?

Thanks in advance.

BB

[yaximik]

Likely PS bonds would block polymerase, but in case of 3'-PS adaptor ends these are out of action as polymerase starts extension at 3'-ends of anchored primers that are not blocked. I have some intermediate adaptors with 4 PS bonds at their 3'-ends, which work in PCR as templates just fine.

[biochembug]

Thanks for the reply, yaximik!

I called on Illumina techsupport on this question. They say there should not be any problem for PCR products with PS bonds to form clusters. Although they did not give me a direct answer, I suspect their small RNA library kit which uses these primers have PS bonds. Therefore the small RNA library may have PS bonds within the final library PCR products.

[yaximik]

Just to make sure we are on the same page. I doubt oligos like 5'-NNNNN*N*N*N-3' can be extended or ligated, so unlikely library contains fragments with internal PS bonds. I could not find any references on that, if someone knows examples please share. Whether internal PS bonds can interfere with polymerase I do not know as I could not find any references either. Oligos with PS bonds at 3'-ends are often used as adaptors producing fragments with terminal PS bonds:

5'-N N N NNN-insert-NNNN*N*N*N-3'
3'-N*N*N*NNN-insert-NNNN N N N-5'

These will not interfere with cluster formation and any PCR amplification with primers that do not have PS at their 3'-ends as terminal PS bonds in library fragments are out of the action locus:

5'-NNNNNNNNNprimerNNNNNNN-3' -----> extension
3'-N*N*N*NNNNNNNNNNNNinsertNNNNNNNNNNNNNNNN.....-5'

[biochembug]

Here is the expected pattern of the ds product (*=PS bonds) in the library:

5'Flowcellbinding-----------*N-Insert----------Flowcellbinding3'
3'Flowcellbinding--------------Insert-N*-------Flowcellbinding5'

To form a cluster, polymerase on the flow-cell has to go through the PS bonds. So my Q was whether this is possible?

[yaximik]

Well, it appears I had incorrect assumption. According to this site

http://https://wikis.utexas.edu/display/GSAF/454+-+all+flavors

454 adaptors have PS bonds on each side. If this is correct these can be ligated. form internal PS bonds and therefore polymerase can get over such bonds in subsequent emPCR aamplifications. Although I could not find confirming references in PubMed, I presume that if Illumina library is created in the same way, then you should not have problems.

[biochembug]

Hmm.. So this corroborates the point made by Illumina techsupport. Thanks yaximik.